Therapeutic constructs for co-delivery of mitotic kinase inhibitor and immune checkpoint inhibitor

ABSTRACT

Disclosed herein are therapeutic constructs including a delivery particle, at least one mitotic kinase inhibitor, and at least one immune checkpoint inhibitor. Also disclosed are therapeutic constructs including a mitotic kinase inhibitor, an immune checkpoint inhibitor, and a chemical linker. These therapeutic constructs cause cancer death by both therapeutic and immune effects and promote targeted delivery of more therapeutics to the surviving cancer cells in a positive feed-back loop. They enhance therapeutic index of free drugs and can be used intratumorally or systemically. This strategy can treat broad cancer types and is particular useful for cancer without obvious receptors for cancer-targeted delivery of otherwise toxic therapeutics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Phase of International Application No.PCT/US2020/041852, filed Jul. 13, 2020; which in turn claims priority toand the benefit of the earlier filing date of U.S. ProvisionalApplication No. 62/873,770, filed on Jul. 12, 2019. Each of theseearlier filed applications is incorporated by reference herein in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant R44CA217534awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE DISCLOSURE

The current disclosure relates to compositions and methods forimmunotherapy treatment. Therapeutic constructs are described based onco-delivery of mitotic kinase inhibitor(s) and immune checkpointinhibitor(s). These therapeutic constructs have greater therapeuticindex and/or trigger adaptive anti-cancer immunity better than the freedrug counterparts for broad cancer treatment.

BACKGROUND OF THE DISCLOSURE

Immune checkpoint inhibitors, such as antibodies against PD-L1, PD-1,and CTLA-4 have shown promising outcomes in clinics, gaining fast trackFDA approval for treating many cancer types. However, while patients whorespond to immune checkpoint blockade may show robust and durableresponses, only a minority of total patients respond, and even forpatients with high PD-L1 expression, response rates remain under 50%(Reck et al., NEJM 375(19):1823-1833, 2016). Furthermore, many initialresponders will develop resistance and ultimately relapse (Jenkins etal., Brit J Canc. 118:9, 2018).

While in general immune checkpoint blockade has less severe and distincttoxicity from chemotherapy, autoimmune disorders caused by immunotherapyis a concern (Tocut et al., Autoimmunity Rev 17(6):610-616, 2018).Systemic distribution of these antibodies can cause aberrant anduncontrolled immune response, leading to immune-related adverse effects(irAEs) (Reynolds et al., J Clin Oncol. 36(16_suppl):3096, 2018). Whilegenerally manageable, discontinuation of treatment due to irAEs haveoccurred and in some instances irAEs can be fatal.

To improve cancer treatment outcomes, studies have investigated the usechemotherapy in combination with immune checkpoint inhibitors. Forinstance, one clinical trial has investigated the combination ofnab-paclitaxel (abraxane) with PD-L1 antibody (atezolizumab) given asfree agents for metastatic TNBC (Schmid et al., N Engl J Med379(22):2108-2021, 2018). For preclinical studies, nanoparticle forco-delivery of docetaxel and PD-L1 antibody (Xu et al., Inter J Nanomed.14:17-32, 2018) or doxorubicin and PD-L1 antibody (Emami et al., MolPharm 16(3):1184-1199, 2019) have been reported. However, co-delivery ofmitotic kinase inhibitor and an immune checkpoint inhibitor has neverbeen reported as free agents or co-delivered on particles or withchemical linkers.

Mitotic kinase inhibitors have single-agent potency to kill cancer cellsby inducing cell cycle arrest and apoptosis. Unlike chemotherapeuticswhich kill any fast dividing cells, mitotic kinase inhibitors areconsidered targeted therapy, and should be more specific to cancer cellsthan chemotherapeutics.

Nevertheless, major limitations of current mitotic kinase inhibitorssuch as PLK1 small molecule inhibitors include low solid tumorbioavailability and toxic side effects to other rapidly dividing cells,particularly to hematopoietic precursor cells (Gjertsen & Schoffski,Leukemia 29(1):11-19, 2015). PLK1 inhibitors need to have longhalf-lives in order to achieve sufficient tumor bioavailability. Thisresults in longer exposure times with hematopoietic precursor cells inblood and bone marrow, which leads to dose-limiting toxicity ofneutropenia (low neutrophils) and thrombocytopenia (low platelets) (deBraud et al., Annals of Oncol./EMSO 26(11):2341-2346, 2015; Schoffski etal., Euro J Canc. 48(2):179-186, 2012; Lin et al., Brit J Canc.110(10):2434-2440, 2014; Frost et al., Curr Oncol. 19(1):e28-35, 2012).This highlights the need for targeted delivery of the mitotic kinaseinhibitors to cancer cells over non-target cells.

Additionally, PLK1 inhibitors can also inhibit other PLK family membersPLK2 and PLK3, which may further lead to toxic side effects (Raab etal., Nat. Comm. 2:395, 2011). Of all PLK1 inhibitors, Volasertib(Boehringer Ingelheim) has shown the most promise having reached phaseIII clinical trial but only for acute myeloid leukemia (blood cancer)(Gjertsen & Schoffski, Leukemia 29(1):11-19, 2015), but results in phaseIII trials were not promising perhaps due to insufficient dosages (e.g.,limited by toxicity). Inhibition of PLK1 for cancer therapy remains aclinical challenge.

Further, previous studies have elucidated the extensive interplay ofPLK1 with many genes that regulate cancer progression and immune evasion(Zitouni et al., Nat Rev Mol Cell Biol 15(7):433-452, 2014; Zhang etal., BMC Cancer 17(1):861, 2017; Liu et al., Translational Oncol.10(1):22-23, 2016; Fu & Wen, Cancers 9(10), 2017), this highlights thatmonotherapy with PLK1 inhibitors alone may be ineffective. Mitotickinase inhibitors alone are also quite toxic as shown in clinical trialsof various PLK1 inhibitors.

SUMMARY OF THE DISCLOSURE

Described herein is development of new therapeutic constructs based onco-delivery of mitotic kinase inhibitor(s) (or mitotic inhibitor(s)) andcheckpoint inhibitor(s). These therapeutic constructs have greatertherapeutic index than the free drug counterparts and are useful forbroad cancer treatment.

Strategies to improve the response, improve therapeutic efficacy, andmanage toxicities of immune checkpoint blockade and mitotic kinaseinhibitors are highly warranted for treating cancers. Single agent(namely, therapeutic constructs) delivery of immune checkpointinhibitors and mitotic kinase inhibitors will co-localize therapeuticeffects to achieve synergy, while reducing systemic toxicities of thedrugs.

Mitotic inhibitors and mitotic kinase inhibitors have single agentpotency to kill cancer cells by inducing cell cycle arrest andapoptosis.

A mechanism by which cancer cells avoid death by mitotic inhibition isto upregulate immune checkpoint to avoid immune-mediated cell killingand hence remain immunologically invisible (FIG. 1A). Thus, by combiningmitotic inhibitors with immune checkpoint inhibitors, cells whichsurvive mitotic inhibitors can be attacked by immune cells (i.e.cytotoxic CD8+ T cells) to generate an immune response (FIG. 1B).

Described herein are engineered particles (therapeutic constructs) forco-delivery of at least one mitotic inhibitor or mitotic kinaseinhibitor and at least one immune checkpoint inhibitor. Data providedherein illustrate how delivery of a mitotic kinase inhibitor along withan immune checkpoint inhibitor on a therapeutic construct can improveefficacy and reduce toxicity (by reducing doses by 5-fold in theillustrative lung metastasis model).

The immune checkpoint inhibitors on the engineered particles not onlyenable T cells to attack the cancer cells, but it also serve as a homingtarget agent to the surviving cancer cells.

Data also indicate that not only can the provided therapeutic constructkill cancer cells, but it can also trigger adaptive antitumor responsethat slow down the development of a distant tumor (e.g., metastasis).

The mitotic kinase inhibitors may be in the class of small moleculeinhibitors, antibody-based drugs, or oligonucleotides (e.g., siRNA,miRNA, antisense oligonucleotide).

The therapeutic constructs can be administered locally or intratumorallyfor instance to readily accessible tumors such as melanoma, head andneck cancer, breast cancer, and lymphoma; or systemically for othercancers such as lung cancer, liver cancer, pancreatic cancer, prostatecancer, brain cancer, kidney cancer, blood cancer, gastric cancer, coloncancer, rare cancer, and metastatic cancer.

Engineered therapeutic constructs can have a diameter in the nanometersor micrometer range, and can be made of any materials (e.g., lipid,organic materials, inorganic materials, polymers, and hybrids orcombinations thereof) capable of loading the therapeutic agents/adjuvantcargos, delivering them to the target sites (cancer cells, immune cells,extracellular matrices, etc.), and allowing them to have the desiredfunctions.

Adjuvant can optionally be co-delivered on the same therapeuticconstruct to boost antitumor T cell repertoire to enhance thetherapeutic effect. Mitotic kinase inhibitors will kill cancer cellsleading to antigen release, adjuvants will simulate danger signals toactivate pattern recognition receptors to stimulate immune cells, andimmune checkpoint inhibitor will remove the brakes applied by the tumorcells on immune cells. In this way, the single agent therapeutic canovercome various strategies by which cancer cells evade the immuneresponse to provide sustained cancer cell killing effects.

Optionally, example therapeutic constructs also contain one or morehoming agents (antibodies, aptamers, ligands, peptides, etc.) thatenable them to be preferentially delivered to and/or taken up by targetcancer cells or various immune cell types (e.g., DCs, macrophages,monocytes, T cells).

The herein provided therapeutic constructs may be used alone or incombination with standard therapeutics, including, but not limited to,chemotherapy, surgery, targeted therapies, and radiation therapy.

Alternatively, other targeted therapeutics (e.g., small moleculeinhibitors or antibodies targeting other oncoproteins, or medicalradioactive isotopes) can be loaded directly on/in the therapeuticconstructs as a therapeutically active agent.

For local delivery, the therapeutic constructs optionally can beformulated into topical or microneedle formulations.

Provided herein are therapeutic constructs that include: a deliverysystem; at least one mitotic inhibitor or mitotic kinase inhibitorcoupled to or contained within the delivery system; and at least oneimmune checkpoint inhibitor coupled to or contained within the deliverysystem. In examples of this embodiment of the therapeutic construct, thedelivery system includes a liposome, a lipid-based particle, a polymericparticle, an inorganic or organic nanoparticle or microparticle, or ahybrid thereof. In particular embodiments of the provided therapeuticconstruct, nanoparticles with a hydrodynamic size of 5 nm to 999 nm(e.g., about 80 nm to about 200 nm, about 90 nm to about 130 nm; or lessthan 150 nm), as measured in an aqueous solution (such as PBS, Trisbuffer, or water) are employed. In yet other examples, the therapeuticconstructs are microparticles with a hydrodynamic size of 1 micron to1000 micron. In some embodiments, the delivery system has a size ofabout 5 nm to about 200 nm, about 5 nm to about 90 nm, about 5 nm toabout 20 nm, about 30 nm to about 100 nm, about 30 nm to about 80 nm,about 30 nm to about 60 nm, about 40 nm to about 80 nm, about 70 nm toabout 90 nm, or about 5 nm, about 10 nm, about 20 nm, about 30 nm, about40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm,or about 100 nm.

In some embodiments, the therapeutic construct further includes anadjuvant. In some embodiments, the therapeutic construct does notinclude a tumor-specific antigen.

Also provided herein are therapeutic constructs that include an immunecheckpoint inhibitor, a mitotic kinase inhibitor, and a chemical linkerlinking the immune checkpoint inhibitor and the mitotic kinaseinhibitors. In some embodiments, the immune checkpoint inhibitor is anoligonucleotide, a polynucleotide, a small molecule inhibitor, or anantibody. In some embodiments, the mitotic kinase inhibitor is anoligonucleotide, a polynucleotide, a small molecule inhibitor, or anantibody. In some embodiments, the therapeutic construct is anantibody-oligonucleotide conjugate, a small molecule-oligonucleotideconjugate, or a small molecule-small molecule conjugate. In someembodiments, the immune checkpoint inhibitor is an antibody (e.g., onethat is against PD-L1, PD-1, TIM-3, LAG-3, or CTLA-4). In someembodiments, the mitotic kinase inhibitor is a small molecule inhibitor,such as an inhibitor of PLK1 (e.g., GSK461364, BI2536, Tak960, NMS-P937,volasertib), Chk 1 kinase (e.g., LY2603618, prexasertib, or AZD7762), aRHA helicase A (e.g. YK-4-279), cyclin-dependent kinase 1/2 (e.g.,AZ703), Aurora kinase A (e.g., alisertib). In some embodiments, themitotic kinase inhibitor is an oligonucleotide, such as a siRNA or anantisense oligonucleotide against the mitotic kinase gene (e.g., siRNAagainst PLK1, such as siPLK1).

Also provided are compositions that include at least one therapeuticconstruct as described herein. Optionally, such compositions furthercomprise at least one pharmaceutically acceptable carrier, excipient, ordiluent.

Another embodiment is a method of treating cancer, which method includesadministering to a subject (such as a human subject) with cancer aneffective amount of a provided therapeutic construct, or a compositioncontaining such a therapeutic construct, to reduce one or more symptomsof the cancer.

Also provided are methods of treating a cell exhibiting symptoms ofcancer including contacting the cell with a therapeutically effectiveamount of a provided therapeutic.

Also provided are methods of treating a cell obtained from a subjectexhibiting symptoms of cancer including contacting the cell with atherapeutically effective amount of a provided therapeutic construct, ora composition containing such a therapeutic construct.

Also provided are methods of treating a cell obtained from a subjectexhibiting symptoms of cancer that include contacting a cell ex vivowith a therapeutically effective amount of a provided therapeuticconstruct, or a composition containing such a therapeutic construct.

In any of the cell-based embodiments, it is contemplated that the cellin some instances is a cancer cell. In other instances, the cell is nota cancer cell. In various embodiments, the cell is an immune cell.Optionally, in any of the cell-based embodiments, the cell may be from ahuman subject, or from another mammalian subject.

Yet another embodiment is a method of treating a subject diagnosed ashaving a hyperproliferative disease or condition, which method includesadministering to the subject an effective amount of a compositionincluding at least one of the provided therapeutic constructs.

Also provided are methods of enhancing effect of an anti-cancer therapyin a subject (such as a human subject) in need thereof, includingadministering to a subject in need thereof: an effective amount of aprovided therapeutic construct, or a composition containing such atherapeutic construct; and at least one anti-cancer agent (e.g., achemotherapeutic agent, targeted therapeutic agent, or an immunecheckpoint inhibitor). Optionally, the therapeutic construct orcomposition and the anti-cancer therapy are administered sequentially orconcurrently.

Yet another embodiment is a method of enhancing radiation therapy effectin a subject (such as a human subject) diagnosed as having a neoplasia,including administering to a subject in need thereof: an effectiveamount of a provided therapeutic construct, or a composition containingsuch a therapeutic construct; and at least one radiation therapy.Optionally, the therapeutic construct or composition and the radiationtherapy are administered sequentially or concurrently.

As used herein, the term “enhancing,” in the context of the therapeuticeffects of an anti-cancer therapy, refers to an increase in thetherapeutic effects of the anti-cancer therapy (e.g., treatment with ananti-cancer agent, radiation therapy, or checkpoint immunotherapy) abovethose normally obtained when the anti-cancer therapy is administeredwithout the therapeutic constructs of the invention. “An increase in thetherapeutic effects” is manifested when there is an acceleration and/orincrease in intensity and/or extent of the therapeutic effects obtainedwith an anti-cancer therapy. It also includes extension of the longevityof therapeutic benefits. It can also manifest where a lower dosage ofthe anti-cancer therapy is required to obtain the same benefits and/oreffects when it is co-administered with the therapeutic constructsprovided by the present invention as when a higher dosage of theanti-cancer therapy is administered alone. The enhancing effectpreferably, but not necessarily, results in treatment of acute symptomsfor which the anti-cancer therapy alone is not effective or is lesseffective therapeutically. Enhancement is achieved when there is atleast a 10% increase (e.g., at least 25%, at least 50%, at least 75%, orat least 100%) in the therapeutic effects when a therapeutic constructof the present invention is co-administered with an anti-cancer therapycompared with administration of the anti-cancer therapy alone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Central hypothesis for therapeutic constructs' activities.(FIG. 1A) Mitotic inhibition (e.g., by PLK1 inhibitor or siRNA) killscancer and releases antigens, but also increases checkpoint (e.g.,PD-L1) expression in the surviving cells, which inhibits anti-cancerimmune response to the surviving cells. (FIG. 1B) Combing mitoticinhibitor and immune checkpoint inhibitor (e.g., on a therapeuticconstruct) leads to synergistic treatment of cancer.

FIGS. 2A-2D. Effects of siRNA against mitotic regulator PLK1 (siPLK1) onnon-small-cell lung carcinoma (NSCLC) cell lines (A549 and H460). (FIG.2A) 48-hr PLK1 mRNA knockdown (HPRT used as house-keeping gene) and(FIG. 2B) 72-hr PLK1 protein reduction at 50 nM siRNA dose. (FIG. 2C)4-day cell viability at 30 nM siRNA dose. (FIG. 2D) Cell cycle arrestincrease in G2/M phase in A549 72 hr post treatment. Antibody(cetuximab) conjugated NP was used to deliver the siPLK1 (C-siPLK1-NP)or scrambled siRNA control (C-siSCR-NP) at 50 nM siRNA BI 2536 (a PLK1inhibitor) was used as a drug benchmark at 10 nM. Data presented asmean±SD from independent duplicates (10,000 events per sample);****P<0.0001 vs. untreat control. Unless specified otherwise, “NP”denotes mesoporous silica nanoparticles coated with cross-linked PEI andPEG, as described in Ngamcherdtrakul et al., Advanced FunctionalMaterials, 25(18):2646-2659, 2015 and U.S. Patent ApplicationPublication No. 2017/0173169.

FIGS. 3A-3C. PLK1 knock-down by siRNA induces PD-L1 expression. (FIG.3A) PLK1 and PD-L1 mRNA expression in A549 (human NSCLC) at 48 hr posttreatment with PLK1 siRNA (siPLK1) or scrambled siRNA (siSCR) normalizedto HPRT housekeeping gene. Data presented as mean±SD from triplicates;****P<0.0001. (FIG. 3B) PD-L1 surface expression of A549 (FIG. 2B) andLLC-JSP (a mouse NSCLC, FIG. 3C) at 72 hr post treatments assessed byflow cytometry (10,000 events per sample). Mouse siPLK1 seq.GUGGGCGUGGUACCAUCUGUU (SEQ ID NO: 1); Human siPLK1 seq.UAUUCAUUCUUCUUGAUCCGG (SEQ ID NO: 2).

FIGS. 4A-4C Treatment effects of mitotic kinase inhibitors on (A) 3-dayviability of LLC-JSP cells, (B) PD-L1 expression levels of survivingcells post treatment with 500 ng/ml of volasertib, alisertib, or AZD7762as determined by flow cytometry and (C) their quantification.

FIGS. 5A-5C. Enhanced cancer treatment with PD-L1 inhibitor and PLK1inhibitor given as free drugs. (FIG. 5A) C57BL/6 mice were injected with200K LLC-JSP cells in right flank. On day 8 post tumor inoculation, micewere grouped (n=7-8) and received i.p. treatments of control vehicles(PBS and HCl/saline), PLK1 inhibitor volasertib (20 mg/kg), mouse PD-L1antibody (200 μg per mouse, BioXCell), or combination of PLK1 inhibitorand PD-L1 antibody. Treatments were administered every 5 days for 3doses. (FIG. 5B) Tumor growth of mice. (FIG. 5C) Kaplan-Meier Survivalcurve. Data presented as mean±SEM; ***P<0.001, ****P<0.0001.

FIGS. 6A-6D Nanoparticle delivery of PLK1 inhibitor volasertib(iPLK1-NP) to mouse NSCLC cells. (FIG. 6A) Schematic of synthesis ofiPLK1-NP. (FIG. 6B) Hydrodynamic size of NP (with no inhibitor) andiPLK1-NP measured with Zetasizer. (FIG. 6C) Viability of LLC-JSP cellstreated with volasertib (in 1% DMSO/PBS), iPLK1-NP (in PBS), or 1%DMSO/PBS for 4 days. Data presented as mean±SD from 4 independentsamples; ****P<0.0001. (FIG. 6D) PD-L1 surface expression of LLC-JSPcells treated with PBS or iPLK1-NP (42 μg/ml NP, 210 ng/ml volasertib)for 3 days.

FIGS. 7A-7E. Nanoparticle for co-delivery of PLK1 inhibitor (iPLK1) andPD-L1 antibody (p-iPLK1-NP). (FIG. 7A) Schematic and (FIG. 7B)hydrodynamic size of p-iPLK1-NP containing 4 wt. % of PD-L1 antibody and0.5 wt. % of PLK1 inhibitor. (FIG. 7C) 5-day cell viability of LLC-JSPcells treated with iPLK1-NP or p-iPLK1-NP. Data presented as mean±SDfrom 4 independent samples; ns—not significant. PD-L1 surface expressionassessed by flow cytometry after LLC-JSP cells were incubated withvarious treatments as specified for (FIG. 7D) 2 hrs and (FIG. 7E) 2days. Doses: free PD-L1 antibody (50 μg/ml), iPLK1-NP (NP containing 50μg/ml volasertib), and p-iPLK1-NP (NP containing 50 μg/ml volasertib and2 μg/ml PD-L1 antibody). Left of FIGS. 7D and 7E: representativehistograms, right: median intensity (RFU). Data presented as mean±SDfrom independent duplicates (10,000 events per sample); *P<0.05,**P<0.01, ***P<0.001, ****P<0.0001. Unless specified otherwise, thepercent loading is by weight of nanoparticle throughout the application.

FIGS. 8A-8E. p-iPLK1-NP elicits anti-tumor immune effects. (FIG. 8A)100K LLC-JSP cells were injected in right flank and 40K cells wereinjected in left flank of C57BL/6 mice. On day 12 post tumorinoculation, mice received intratumoral treatments of saline, p-NP,iPLK1-NP, or p-iPLK1-NP to the right (local) tumor. 0.5 mg NP containing4 wt. % of PD-L1 antibody and 0.5 wt. % of PLK1 inhibitor in 50 μl perdose for 3 doses total. (FIG. 8B) Local tumor growth. (FIG. 8C) Distant(untreated) tumor growth of individual mice. (FIG. 8D) Kaplan MeierSurvival curve. (FIG. 8E) Mice were injected with tumors as described inFIG. 8A and received treatments of saline or p-iPLK1-NP. One day afterlast treatment, tumors were harvested to assess tumor infiltratinglymphocytes (TILs) with flow (50,000 events per sample). Data presentedas mean±SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

FIGS. 9A-9C. p-iPLK1-NP improves survival of mice bearing metastaticlung tumors. (FIG. 9A) C57BL/6 mice were injected with 200K LLC-JSPcells intravenously, which created tumors in the lungs. After 3 days,mice were randomly assigned systemic treatments of saline, free drugs(12.5 μg volasertib and 100 μg PD-L1 antibody), or p-iPLK1-NP(containing 2.5 μg volasertib and 20 μg PD-L1) for a total of 4 doses.(FIG. 9B) Kaplan-Meier Survival curve. *P<0.05, **P<0.01 (Log-rankMantel-Cox test). (FIG. 9C) Mice weight change post first treatment.

FIG. 10. Efficacy of p-iPLK1-NP is dependent on CD8+ T cells. C57BL/6mice were injected with 200K LLC-JSP cells intravenously. After 3 days,mice were treated with saline, p-iPLK1-NP (i.v., containing 2.5 μgvolasertib and 20 μg PD-L1), or p-iPLK1-NP+anti-CD8 (200 μg twiceweekly). (A) Kaplan-Meier Survival curve. *P<0.05, **P<0.01, ***P<0.001(Log-rank Mantel-Cox test).

FIGS. 11A-11C. Targeting and treatment specificity of p-iPLK1-NP. (A)PD-L1 expression of 4T1 cells 4-day post treatment of p-iPLK1-NP. Cells(control and p-iPLK1-NP treated) were harvested and incubated withp-iPLK1-NP tagged with dye-siRNA for 1 hr. (B) Cellular uptake ofp-iPLK1-NP. (C) Cell viability of murine cancer cells (LLC-JSP, 4T1,B16-F10) and murine bone marrow-derived dendritic cells (BMDC) treatedwith p-iPLK1-NP.

FIGS. 12A and 12B. Inhibition of PLK1 reduces phosphorylation of STAT3.Western blot showing protein expression of PLK1, Pl3Ka, phosphorylatedSTAT3 (Tyr705), phosphorylated AKT (Ser473), and p-Actin 3 days posttreatment (50 nM siRNA) in A549 and H460 NSCLC cell lines. FIG. 12Bshows that NP can also deliver siRNA against PD-L1 (siPDL1) resulting ineffective knock down of PD-L1 protein expression (as measured by flowcytometry) in LLC-JSP cells. The cells were treated with NP containing30 nM siRNA against PD-L1 (siPDL1) or 30 nM scrambled siRNA (siSCR) at 2wt. % siRNA. At 72 hr post treatment, cells were harvested and assessedfor PD-L1 protein expression by flow cytometry. RFU=Relativefluorescence units.

FIG. 13. Adding CpG to p-iPLK1-NP enhances therapeutic benefit asdemonstrated by Kaplan Meier Survival curve. 100K LLC-JSP cells wereinjected in right flank and 40K cells were injected in left flank ofC57BL/6 mice. On day 12 post tumor inoculation, mice receivedintratumoral treatments of saline, p-NP, iPLK1-NP, p-iPLK1-NP, orp-iPLK1-NP-CpG to the right (local) tumor. 0.5 mg NP (2.5 μg iPLK1, 20μg PD-L1 antibody, 20 μg CpG) in 50 μl was administered every 3 days fora total of 3 doses.

FIGS. 14A-14C. Antibody-drug conjugate (ADC) of alisertib (Aurora KinaseA inhibitor) and PD-L1 antibody. (A) Synthesis scheme of PD-L1-antibodyalisertib conjugate (ADC). (B) Treatment effect of ADC versus freealisertib of equivalent dose on viability of LLC-JSP cells (2-days).Free alisertib was dissolved in DMSO before use. (C) Effect of PD-L1 onthe viability of LLC-JSP cells.

FIG. 15. Topical siRNA-NP in pig skin with and without microneedleroller pre-treatment. Fluorescent images of pig skin treated with onetopical application of Dy677-siSCR-NP in Aquaphor for one hour with andwithout pre-treating skin with a microneedle roller. siRNA signal isnoted with arrows. Tissues were also stained for nuclei with Hoechst33342.

FIG. 16. Topical siRNA-NP/Tween-Aquaphor in mice with and withoutmicroneedle roller pre-treatment. Fluorescent images of mouse skintreated with one topical application of Dy677-siSCR-NP in Tween/Aquaphorfor 1.5 hour with and without pre-treating skin with a microneedleroller. siRNA signal is noted with arrows. Tissues were also stained fornuclei with Hoechst 33342.

FIGS. 17A and 17B. EGFR knock down efficacy of topical siRNA-NP withmicroneedle roller versus injected siRNA-NP. Mouse skin was harvested at3 days after one topical treatment with siEGFR-NP or siSCR-NP inTween/Aquaphor with microneedle roller application (A) or 3 days afterone injection of siEGFR-NP or siSCR-NP in saline (B). Skin tissue wasfixed and stained with fluorescently labelled EGFR antibody for EGFRsignal quantification. 4-8 images (20×) were processed per condition and3 animals per group.

FIG. 18. Dextran-based microneedle containing NP loaded withDy677-siRNA.

REFERENCE TO SEQUENCE LISTING

The nucleic acid sequences described herein are shown using standardletter abbreviations for nucleotide bases, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included in embodiments where itwould be appropriate. A computer readable text file, entitled“2LO5988.txt” created on or about Nov. 30, 2021, with a file size of 4KB, contains the sequence listing for this application and is herebyincorporated b reference in its entirety.

SEQ ID NO: 1 is a Mouse siPLK1 sequence:  GUGGGCGUGGUACCAUCUGUUSEQ ID NO: 2 is a Human siPLK1 sequence:  UAUUCAUUCUUCUUGAUCCGGSEQ ID NO: 3 is a scrambled siSCR sequence: UUAGUCGACAUGUAAACCA

DETAILED DESCRIPTION

The herein described therapeutic approach for cancer treatment utilizesengineered particles or chemical linkers for co-delivery of mitotickinase inhibitors and immune checkpoint inhibitors to create therapeuticconstructs that localize both classes of drugs in the same cells forcancer therapy.

Upon intratumoral or systemic administration of a provided therapeuticconstruct to cancer cells, the mitotic kinase inhibitors will put cancerto cell cycle arrest, leading to programmed cell death, and increasedimmune checkpoint expression (e.g., PD-L1) of the surviving cancercells. Therefore, immune checkpoint inhibitors (e.g., antibodies againstPD-L1) will enhance targeted delivery of the construct to the survivingcells as well as enable cytotoxic T cells to attack the cancer.

Since mitotic kinase inhibitors can upregulate PD-L1 receptors, thisstrategy can treat broad cancer types and is particular useful forcancer without obvious receptors for targeted delivery of otherwisetoxic therapeutics such as mitotic kinase inhibitors.

Death of cancer cells also releases tumor antigens and together withcheckpoint inhibition can trigger adaptive immunity to attack cancer orprevent cancer spread or relapse. Optionally, an adjuvant can be addedto the therapeutic construct to increase the antitumor immune response.

The invention utilizes new discovery cancer biology and immunology, andengineered particles to create new drug candidates to increase efficacywhile reduce toxicity compared to free drug counterparts.

In certain embodiments, the delivery vehicle includes a MSNP core (e.g.,˜50 nm) for drug loading, coated with a bioreducible cross-linkedcationic polymer, e.g., polyethyleneimine (PEI) for oligo loading andendosomal escape; and a stabilizer, e.g., polyethylene glycol (PEG),which prevents nanoparticle aggregation, protects oligo cargos fromdegradation by blood enzymes (Ngamcherdtrakul et al., AdvancedFunctional Materials, 25(18):2646-2659, 2015), and shields the charge ofPEI, enhancing safety. Oligo (siRNA and/or CpG) is loaded last on theconstruct with a few minutes (e.g., 5 minutes) mixing in PBS at roomtemperature; it electrostatically binds to PEI in an oligosequence-independent manner and is protected under the PEG layer fromenzymatic degradation (Ngamcherdtrakul et al., Advanced FunctionalMaterials, 25(18):2646-2659, 2015). The resulting nanoparticle (NP) washighly optimized for siRNA delivery efficacy in terms of MSNP sizes, PEIand PEG molecular weights and compositions, PEI crosslinking conditions(to enhance buffering capacity and lower charge), oligo and (optionally)antibody loadings (Ngamcherdtrakul et al., Advanced FunctionalMaterials, 25(18):2646-2659, 2015). This embodiment of the siRNA-NP hasa rigid MSNP core size (by TEM) of 50 nm and hydrodynamic size (NP withpolymer coatings) of 100 nm with a narrow size distribution. It consistsof 13.5 wt. % PEI, 18.2 wt. % PEG, and can load 2-4 wt. % siRNA or up to10 wt. % of CpG oligo. Drug (e.g., taxane) can be loaded in the MSNPcore or on the polymers at 0.5-3 wt. %. All values in this paragraph areby weight of the nanoparticle. See also US Patent Publication2017/0173169.

In certain embodiments, the immune checkpoint inhibitor is anoligonucleotide, a polynucleotide, a small molecule inhibitor, or anantibody. In some embodiments, the mitotic kinase inhibitor is anoligonucleotide, a polynucleotide, a small molecule inhibitor, or anantibody. In some embodiments, the therapeutic construct is anantibody-oligonucleotide conjugate (Wiener, J. et al. ScientificReports, 10, 1457, 2020), a small molecule-oligonucleotide conjugate(Winkler J., Therapeutic delivery, 4(7), 791-809, 2013), or a smallmolecule-small molecule conjugate.

Provided herein are therapeutic constructs that include: a deliverysystem; at least one mitotic inhibitor or mitotic kinase inhibitorcoupled to or contained within the delivery system; and at least oneimmune checkpoint inhibitor coupled to or contained within the deliverysystem. In examples of this embodiment of the therapeutic construct, thedelivery system includes a liposome, a lipid-based particle, a polymericparticle, an inorganic or organic nanoparticle or microparticle, or ahybrid thereof. For instance, in various examples the delivery vehicleincludes one or more of fullerenes, endohedral metallofullerenes,trimetallic nitride templated endohedral metallofullerenes,single-walled and multi-walled carbon nanotubes, branched and dendriticcarbon nanotubes, gold nanorods, silver nanorods, single-walled andmulti-walled boron/nitrate nanotubes, carbon nanotube peapods, carbonnanohorns, carbon nanohorn peapods, liposomes, nanoshells, calciumphosphate, dendrimers, microparticles, quantum dots, superparamagneticnanoparticles, nanorods, cellulose nanoparticles, silicon, silica andpolymer micro- and nano-spheres, silica-shells, biodegradable PLGAmicro- and nano-spheres, gold nanoparticles, cerium oxide particles,zinc oxide particles, silver nanoparticles, carbon nanoparticles, ironnanoparticles, and/or modified micelles.

In examples of the provided therapeutic construct embodiments, themitotic kinase inhibitor and/or immune checkpoint inhibitor includes anoligonucleotide (e.g., a siRNA or an antisense oligonucleotide), apolynucleotide, a small molecule inhibitor, or an antibody.

In examples of the therapeutic construct, the mitotic kinase inhibitorincludes an inhibitor of at least one of a polo-like kinase (PLK), anAurora kinase, cyclin-dependent kinase (CDK)1, CDK2, HASPIN, monopolarspindle 1 kinase (Mps1), or a NimA-related kinase (NEK). In variousembodiments, the mitotic kinase inhibitor includes one or more ofGSK461364, BI2536, Tak960, NMS-P937, B16727 (volasertib), Chk 1 KinaseInhibitor LY2603618, prexasertib, AZD7762, AU14022, YK-4-279, or AZ703.

In various embodiments, the mitotic inhibitor includes one or more ofetoposide, vinorelbine, mitoxantrone, doxorubicin, estramustine,carboplatin, vinblastine, docetaxel, paclitaxel, and cabazitaxel.

In various embodiments, the immune checkpoint inhibitor includes asiRNA, inhibitor, or antibody against one or more of PD-L1, PD-1, TIM-3,LAG-3, or CTLA-4. By way of example, the therapeutic agent is an immunecheckpoint inhibitor selected from an antibody against PD-L1, PD-1, orCTLA-4. In yet more examples, the immune checkpoint inhibitor includesat least one of: nivolumab, pembrolizumab, ipilimumab, tremelimumab,atezolizumab, avelumab, durvalumab, cemiplimab, pidilizumab, orspartalizumab (PDR001).

The therapeutic constructs provided herein may optionally furtherinclude an adjuvant. It is specifically contemplated that exampleadjuvants used with the provided therapeutic constructs exhibitimmunostimulatory activity. By way of example, an adjuvant useful inembodiments of the provided therapeutic constructs includes one or moreof a CpG oligonucleotide, a DNA TLR agonist containing a CpG sequence, anon-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, ananti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLRagonist, an oil- or surfactant-based adjuvant, a lipopolysaccharide, aplant extract, or a derivative thereof. In specific examples, theadjuvant compound includes a CpG oligonucleotide, imiquimod, resiquimod,gardiquimod, poly IC, poly ICLC, dSLIM, or EnanDIM.

In some embodiments, the therapeutic construct does not include atumor-specific antigen.

Also provided are compositions that include at least one therapeuticconstruct as described herein. Optionally, such compositions furthercomprise at least one pharmaceutically acceptable carrier, excipient, ordiluent.

Another embodiment is a method of treating cancer, which method includesadministering to a subject (such as a human subject) with cancer aneffective amount of a provided therapeutic construct, or a compositioncontaining such a therapeutic construct, to reduce one or more symptomsof the cancer.

Also provided are methods of treating a cell exhibiting symptoms ofcancer including contacting the cell with a therapeutically effectiveamount of a provided therapeutic.

Also provided are methods of treating a cell obtained from a subjectexhibiting symptoms of cancer including contacting the cell with atherapeutically effective amount of a provided therapeutic construct, ora composition containing such a therapeutic construct.

Also provided are methods that include contacting a cell ex vivo with atherapeutically effective amount of a provided therapeutic construct, ora composition containing such a therapeutic construct.

In any of the cell-based embodiments, it is contemplated that the cellin some instances is a cancer cell. In other instances, the cell is nota cancer cell. In various embodiments, the cell is an immune cell.Optionally, in any of the cell-based embodiments, the cell may be from ahuman subject, or from another mammalian subject.

Yet another embodiment is a method of treating a subject diagnosed ashaving a hyperproliferative disease or condition, which method includesadministering to the subject an effective amount of a compositionincluding at least one of the provided therapeutic constructs. Invarious examples of this embodiment, the hyperproliferative diseaseincludes one or more of cancer, precancer, or cancer metastasis. Inexamples of these methods, the hyperproliferative disease includes oneor more of melanoma, lung cancer, breast cancer, pancreatic cancer,brain cancer, prostate cancer, head and neck cancer, kidney cancer,colorectal cancer, lymphoma, colon cancer, or liver cancer.

In any of the provided methods of treating a subject, it is contemplatedthat administration can be by a variety of methods. For instance, inexamples of treatment methods, administering includes one or more of:injection to or at a tumor in the subject; infusion locally to or at atumor in the subject; systemic injection in the subject; systemicinfusion in the subject; or topical application to the subject. In otherexamples, administering includes microneedle application.

Also provided are methods of enhancing effect of an anti-cancer therapyin a subject (such as a human subject) in need thereof, includingadministering to a subject in need thereof: an effective amount of aprovided therapeutic construct, or a composition containing such atherapeutic construct; and at least one anti-cancer agent (e.g., achemotherapeutic agent, a targeted therapeutic agent, or an immunecheckpoint inhibitors). Optionally, the therapeutic construct orcomposition and the anti-cancer therapy are administered sequentially orconcurrently.

Yet another embodiment is a method of enhancing radiation therapy effectin a subject (such as a human subject) diagnosed as having a neoplasia,including administering to a subject in need thereof: an effectiveamount of a provided therapeutic construct, or a composition containingsuch a therapeutic construct; and at least one radiation therapy.Optionally, the therapeutic construct or composition and the radiationtherapy are administered sequentially or concurrently.

Also provided herein is a kit including an therapeutic constructdescribed herein and at least one anti-cancer agent. In someembodiments, the anti-cancer agent is a chemotherapeutic agent, atargeted therapeutic agent, or an immune check point inhibitor.

Aspects of the disclosure are now described with additional detail andoptions to support the teachings of the disclosure, as follows: (I)Therapeutic Constructs; (II) Mitotic Kinases and Inhibitors Thereof;(III) Immune Checkpoint Inhibitors; (IV) Optional AdditionalComponent(s); (V) Delivery Systems; (VI) Antibodies; (VII)Pharmaceutical Compositions and Administration Formulations; (VIII)Exemplary Methods of Use; (IX) Kits; (X) Exemplary Embodiments; and (XI)Examples.

(I) THERAPEUTIC CONSTRUCTS

Described herein is a new class of therapeutics (generally, “therapeuticconstructs”) that include an engineered particle which co-delivers atleast two active agents, which include at least one mitotic kinaseinhibitor and at least one immune checkpoint inhibitor, to cancer cells.Also disclosed herein are therapeutic constructs that include an immunecheckpoint inhibitor, a mitotic kinase inhibitor, and a chemical linkerconnecting the two (an antibody-drug conjugate), e.g., such as any ofthose described herein. The ratio of active agents (e.g., mitotic kinaseinhibitor to immune checkpoint inhibitor or immune checkpoint inhibitorto mitotic kinase inhibitor) can be, e.g., about 1-20 (e.g., about 2-8,about 4-6, about 2, about 4, or about 6). The mitotic kinase inhibitorcan be present at 0.01 wt. % to 99.9 wt. % of the therapeutic construct(e.g., 0.01 to 1 wt. %, 1 to 5 wt. %, 1 to 10 wt. %, 1 to 20 wt. %, 10to 30 wt. %, 10 to 40 wt. %, 10 to 50 wt. %, 25 to 75 wt. %, 40 to 60wt. %, 50 to 75 wt. %, 50 to 80 wt. %, 75 to 90 wt. %, 75 to 95 wt. %,or 75 to 99.9 wt. %), and the immune checkpoint inhibitor can be presentat 0.01 wt. % to 99.9 wt. % (e.g., 0.01 to 1 wt. %, 1 to 5 wt. %, 1 to10 wt. %, 1 to 20 wt. %, 10 to 30 wt. %, 10 to 40 wt. %, 10 to 50 wt. %,25 to 75 wt. %, 40 to 60 wt. %, 50 to 75 wt. %, 50 to 80 wt. %, 75 to 90wt. %, 75 to 95 wt. %, or 75 to 99.9 wt. %). These therapeuticconstructs reduce the doses required to achieve the efficacy by, e.g.,about five-fold, allowing the drugs to be given together withoutreaching their dose-limiting toxicity. They create adaptive immunitythat enhances tumor inhibition and development at local (treated) anddistant (non-treated) sites (e.g., metastasis), and survival of thetreated subject. Once treated with the therapeutic constructs, cancerundergoes programmed cell death, while the surviving cells overexpressimmune checkpoint molecules such as PD-L1. This enables more targeteddelivery of the constructs to the remaining cancers, that otherwise maynot have significant expression of receptors for targeted delivery, in afeed-forward manner. The therapeutic constructs are also applicable tobroad cancer types since mitotic kinases are found in all cancers, whichwould overexpress immune checkpoint molecules such as PD-L1 upon mitotickinase inhibition.

In some examples, the chemical linker may include one or more of ahydrazine; a disulfide; N-succinimidyl-4-(2-pyridyldithio)butanoate;N-succinimidyl-4-(2-pyridyldithio)-2-sulfo butanoate; perfluorophenyl3-(pyridin-2-yldisulfanyl)propanoate; 2,5-dioxopyrrolidin-1-yl3-methyl-3-(pyridin-2-yldisulfanyl)butanoate; Gly-Phe-Leu-Gly;Ala-Leu-Ala-Leu; Val-Cit; Phe-Lys; Val-Ala; Ala-Phe-Lys; Phe-Lys;(Gly)_(n), wherein n is 1-20; a β-glucuronide linker; maleimidocaproyl;N-(maleimidomethyl)cyclohexane-1-carboxylate;4-(4-acetylphenoxy)butanoic acid; dibromomaleimide; para-aminobenzoicacid; 4-nitrophenol; acetic acid; formic acid; 4-maleimidobutyric acidN-succinimidyl ester; N-(4-maleimidobutyryloxy)succinimide;N-(6-maleimidocaproyloxy)succinimide; 3-maleimidopropionic acidN-succinimidyl ester; N-(3-maleimidopropionyloxy)succinimide;5-maleimidovalericacid-NHS; linear, branched, or multi-arm polyethyleneglycol having a molecular weight of 100-10000 Da;propargyl-N-hydroxysuccinimidyl ester; pyrophosphate;succimimidyl-4-azidobutyrate; 4-azidobenzoic acid N-hydroxysuccinimideester; tert-butyl1-(4-formylphenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-oate; or aresidue thereof. In some embodiments, the chemical linker includesN-(maleimidomethyl)cyclohexane-1-carboxylate or a residue thereof (e.g.,sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Insome embodiments, the chemical linker includes a polyethyleneglycol(e.g. a linear polyethyleneglycol) having a molecular weight of100-10000 Da or a residue thereof.

This strategy will have many key features; they are efficacious, safedue to lower doses needed (vs. free drug counterparts), durable becausethey train and harness body immune cells to attack cancer with memoryeffects, applicable to many types of cancer, and can be given bothlocally for easily accessible tumors and systemically for deeper tumorsand metastatic tumors.

It will be understood that the amount of each component in a therapeuticconstruct (for instance, a mitotic inhibitor, a mitotic kinaseinhibitor, an immune checkpoint inhibitor, the delivery vehicle, or anycomponent of the delivery vehicle) may vary, depending in theembodiment. By way of example, any individual component may make up0.001% to 80% by weight, 0.01% to 75% by weight, 0.5 to 50% by weight,0.5 to 10% by weight, 0.5 to 5% by weight, 1 to 10% by weight, or 2 to4% by weight, of the therapeutic construct.

(II) MITOTIC KINASES AND INHIBITORS THEREOF

Cancer is characterized by uncontrolled cell reproduction. Mitosis is astage in the cell cycle during which a series of complex events ensurethe fidelity of chromosome separation into two daughter cells. Severalcurrent cancer therapies, including the taxanes and vinca alkaloids, actto inhibit the mitotic machinery. Mitotic progression is largelyregulated by proteolysis and by phosphorylation events that are mediatedby mitotic kinases. Aurora kinase family members (e.g., Aurora A, AuroraB, Aurora C) regulate mitotic progression through modulation ofcentrosome separation, spindle dynamics, spindle assembly checkpoint,chromosome alignment, and cytokinesis (Dutertre et al. Oncogene 21:6175, 2002; Berdnik et al. Curr. Biol. 12: 640, 2002). Overexpressionand/or amplification of Aurora kinases have been linked to oncogenesisin several tumor types including those of colon and breast (Warner etal. Mol. Cancer Ther. 2: 589, 2003; Bischoff et al. EMBO 17: 3062, 1998;Sen et al. Cancer Res. 94: 1320, 2002). Moreover, Aurora kinaseinhibition in tumor cells results in mitotic arrest and apoptosis,suggesting that these kinases are important targets for cancer therapy(Ditchfield, J. Cell Biol. 161: 267, 2003; Harrington et al. Nat Med10(3): 262-267, 2004).

Mitotic Kinases: In particular embodiments, mitotic kinases includekinases in the Aurora family of serine/threonine kinases essential forcell proliferation (Bischoff & Plowman, Trends in Cell Biology 9:454-459, 1999; Giet & Prigent, J Cell Science 112: 3591-3601, 1999;Nigg, Nat. Rev. Mol. Cell Biol. 2: 21-32, 2001; Adams et al., Trends inCell Biology 11: 49-54, 2001). Since its discovery in 1997 the mammalianAurora kinase family has been closely linked to tumorigenesis. The mostcompelling evidence for this is that overexpression of Aurora-Atransforms rodent fibroblasts (Bischoff et al., EMBO J. 17: 3052-3065,1998). Inhibitors of the Aurora kinase family therefore have thepotential to block growth of all tumor types.

The three known mammalian family members, Aurora-A (“1”), B (“2”) and C(“3”), are highly homologous proteins responsible for chromosomesegregation, mitotic spindle function and cytokinesis. They are highlyconserved in the C-terminal region, where the kinase domain is located,and show sequence differences in the N-terminal domain (Nat. Rev.Cancer, 5: 42-49, 2005). Aurora expression is low or undetectable inresting cells, with expression and activity peaking during the G2 andmitotic phases in cycling cells. In mammalian cells proposed substratesfor Aurora include histone H3, a protein involved in chromosomecondensation, and CENP-A, myosin II regulatory light chain, proteinphosphatase 1, TPX2, all of which are required for cell division.

Aurora B is expressed between the late G2-phase and telophase. It islocated in the inner centromere region and in the spindle middle zone.It regulates the orientation of the chromosomes at the metaphase plateand corrects wrong kinetochore-microtubule interactions. Itphosphorylates histone H3, which allows the histone to interact with theDNA. This is important for the following chromosome condensation. AuroraC shows high sequence homologies with Aurora B and has functions in themeiosis.

As used herein, the term “Aurora A kinase” refers to a serine/threoninekinase involved in mitotic progression. Aurora A kinase is also known asAIK, ARK1, AURA, BTAK, STK6, STK7, STK15, AURORA2, MGC34538, and AURKA.A variety of cellular proteins that play a role in cell division aresubstrates for phosphorylation by the Aurora A kinase enzyme, including,TPX-2, XIEg5 (in Xenopus), and D-TACC (in Drosophila). The Aurora Akinase enzyme is also itself a substrate for autophosphorylation, e.g.,at Thr288. In some instances, the Aurora A kinase is a human Aurora Akinase.

In particular embodiments, mitotic kinases include Polo-like kinases(“PLKs”). PLKs, including polo-like kinase 1 (“PLK1”), polo-like kinase2 (“PLK2”), polo-like kinase 3 (“PLK3”) and polo-like kinase 4 (“PLK4”),are involved in the formation and changes in the mitotic spindle and inthe activation of CDK/cyclin complexes during mitosis (Strebhardt &Ullrich, Nature Reviews Cancer 6(4): 321, 2006). Plks are overexpressedin tumors, and the overexpression is associated with a poor prognosisand lower overall survival. Therefore, inhibitors of PLKs have beendeveloped as cancer drug therapies.

In particular embodiments, mitotic kinases include cyclin-dependentprotein kinases (CDKs). CDKs are regulators of the timing andcoordination of eukaryotic cell cycle events (Norbury & Nurse, Annu.Rev. Biochem. 61: 441-470, 1992; Sher, Science 274: 1672-1677, 1996). Assuch, CDKs, their regulators, and their substrates are the targets ofgenetic alterations in many human cancers (Kamb et al., Science 264:436-440, 1994; Nobori et al., Nature 368: 753-756, 1994; Spruck et al.,Nature 370: 183-184, 1994; Hunter & Pines, Cell 66: 1071-1074, 1991;Keyomarsi & Pardee, Proc. Natl. Acad. Sci. U.S.A. 90: 1112-1116, 1993;Wang, Nature 369: 669-671, 1994). Members of the cyclin dependent kinasefamily include Cdk2 and Cdk4. Both are active in the G1 phase of cellcycle and regulate entry into the G1/S phase transition. In one pathway,these kinases regulate the phosphorylation of the retinoblastomaprotein. Substrate phosphorylation releases the E2F transcription factorwhich in turn regulates the expression of genes required for S phaseentry. Inhibition of these kinases, therefore, blocks cell entry intothe S phase and downstream proliferative events.

In particular embodiments, mitotic kinases include monopolar spindle 1(MPS1) kinase. MPS1 kinase, also known as TTK, is a dualserine/threonine kinase that controls chromosome alignment andinfluences the stability of the kinetochore-microtubule interaction as akey regulator of the spindle assembly checkpoint (SAC). SAC is essentialfor proper chromosomal alignment and segregation. MPS1 is expressed onlyin proliferating cells and is activated upon phosphorylation duringmitosis, where it is required for proper kinetochore recruitment ofessential SAC proteins such as Mad1 (mitotic arrest deficient protein 1)and Mad2 (mitotic arrest deficient protein 2). MPS1 is alsooverexpressed in a wide range of human tumors and is necessary for tumorcell proliferation.

In particular embodiments, mitotic kinases include Nek ((never inmitosis gene a)-related kinase) 2. Nek2 is a serine/threonine kinasethat localizes to the centrosome and regulates spindle pole organizationand separation through phosphorylation of substrates including C-Napl(nucleosome assembly protein-1), rootletin, and NIp (ninein-likeprotein). In addition to its centrosomal role, Nek2 has also beenimplicated in chromatin condensation and spindle checkpoint control.Nek2 expression and activity are tightly regulated in a cell cycledependent manner. Expression levels are low in G1 and increased in S/G2.Nek2 is abnormally expressed in cancer cells.

In particular embodiments, mitotic kinases include Wee1 kinase. Wee1kinase is a mitotic inhibitor and maintains G2-cell-cycle checkpointarrest for pre-mitotic DNA repair. Wee1 is overexpressed in cancers suchas advanced hepatocellular carcinoma, breast cancer, colon cancer, lungcarcinoma, seminoma, and glioblastoma, and its expression correlatedwith patient survival in mantle cell lymphoma.

One of ordinary skill in the art will understand how to accessrepresentative sequences for mitotic kinases, which are readilyavailable in public sequence databases. The following table providessample sequence information:

Gene Abbreviation Full gene name Representative GenBank Accession #sPLK1 Polo-like kinase 1 NM_005030.5 PLK2 Polo-like kinase 2NM_001252226.1; NM_006622.3 PLK3 Polo-like kinase 3 NM_004073.3;XR_246234.4 PLK4 Polo-like kinase 4 NM_001190799.1; NM_001190801.1NM_014264.4; XM_005262701.2 XM_017007662.1; XM_017007663.1 CDK1Cyclin-dependent kinase 1 NM_001170406.1; NM_001170407.1 NM_001320918.1;NM_001786.4 NM_033379.4; XM_005270303.3 CDK2 Cyclin-dependent kinase 2NM_001290230.1; NM_001798.4 NM_052827.3; XM_011537732.1 CHK1 Checkpointkinase 1 NM_001114121.2; NM_001114122.2 NM_001244846.1; NM_001274.5NM_001330427.1; NM_001330428.1 XM_011542560.2; XM_011542562.2XM_017017146.1; NR_045204.1 NR_045205.1 CHK2 Checkpoint kinase 2NM_001005735.1; NM_001257387.1 NM_001349956.1; NM_007194.3 NM_145862.2;XM_006724114.3 XM_006724116.2; XM_011529839.2 XM_011529840.2;XM_011529841.1 XM_011529842.2; XM_011529844.2 XM_011529845.2;XM_017028560.1 XM_017028561.1; XR_937805.2 XR_937806.2; XR_937807.2 BUB1budding uninhibited by NM_001278616.1; NM_001278617.1 benzimidazole 1NM_004336.4; XR_923001.2 BUBR1 budding uninhibited by NM_001211.5benzimidazole-related 1 MPS1 Monopolar spindle 1 kinase NM_001039396.1NEK2 NIMA related kinase 2 NM_001204182.1; NM_001204183.1 NM_002497.3;XM_005273147.1 HASPIN Histone H3 Associated NM_031965.2 Protein Kinase

Mitotic Kinase Inhibitors: Examples of mitotic kinase inhibitors includeinhibitors for PLK1 (e.g., GSK461364, BI2536, Tak960, NMS-P937, B16727or volasertib), PLK2, PLK3, PLK4, Aurora kinases 1/2 (e.g., alisertib),CDK1/2, CHK1/2 (e.g., AZD7762, prexasertib), BUB1, BUBR1, MPS1, NEK2,HASPIN (Schmit et al., Mol Cancer Ther. 6(7):1920-31, 2007). Thesemitotic kinases can be targeted with small molecule inhibitors,oligonucleotides (e.g., siRNA, miRNA, antisense oligonucleotides),and/or antibodies, all are contemplated in this application.

Non-specific Aurora A inhibitors include: MLN8054 (MillenniumPharmaceuticals, Cambridge, Mass.; Jones et al., Proc Am Soc Clin OncolAnnu Meet 25: 3577, 2007); MK-0457 (VX-680; Harrington et al., Nat Med10(3): 262-267, 2004); SU6668 (Sugen; Lapenna & Giordano, Nature RevDrug Discovery 8: 547-566, 2009, and supplementary information); andZM447439, an inhibitor based on the quinazoline scaffold (Girdler etal., J. Cell Sci., 119, 3664-3675, 2006).

In particular embodiments, selective inhibitors of Aurora A kinaseinclude: compounds disclosed in, for example, US 2008/0045501, U.S. Pat.No. 7,572,784, WO 2005/111039, WO 2008/021038, U.S. Pat. No. 7,718,648,WO 2008/063525, US 2008/0167292, U.S. Pat. No. 8,026,246, WO2010/134965, US 2010/0310651, WO 2011/014248, US 2011/0039826, and US2011/0245234; sodium4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-(i][2]benzazepin-2-yl]amino}-2-methoxybenzoate;KW-2449 (Kyowa Hakko), ENMD-2076 (ENMD-981693; EntreMed); and MK-5108(Vertex/Merck).

Other Aurora kinase inhibitors include: Hesperadin (Hauf et al., J CellBiol 161(2): 281-294, 2003), AZD1152 (quinazoline prodrug, activemetabolite is AZD-1152-HQPA; AstraZeneca, Cambridge, UK; Schellens etal., J Clin Oncol 24:122s, 2006; Yang et al., Blood 110(6): 2034-2040,2007), MLN8237 (Alisertib, selective, competitive, and reversiblesmall-molecule inhibitor of Aurora A kinase; Millennium Pharmaceuticals,Cambridge, Mass.; Gorgun et al., Blood 115(25): 5202-5213, 2010;Friedberg et al., J Clin Oncol 32(1): 44-50, 2014); CYC-116 (Cyclapolin1; Cyclacel Ltd., Cambridge, UK; Taylor & Peters, Curr Opin Cell Biol20: 77-84, 2008); AS-703569 (R-763; Rigel Pharmaceuticals, SanFrancisco, Calif.); AT9283 (Astex; Howard et al., J Med Chem 52(2):379-388, 2009); PHA-739358 (3-aminopyrazole derivative; Nerviano MedicalSciences; Carpinelli et al., Mol Cancer Ther 6(12): 3158-3168, 2007);PHA-680632 (Soncini et al., Clin Cancer Res 12(13): 4080-4089, 2006);SNS-314 (Sunesis Pharmaceuticals, San Francisco, Calif.; Lapenna &Giordano, Nature Reviews Drug Discovery 8: 547-566, 2009, andsupplementary information); and PF-3814735 (Bhattacharya et al., AmAssoc Canc Res 68(9) Supplement LB-147, 2008). Reviewed in Gautschi etal., Clin. Cancer Res. 14(6): 1639-48, 2008. WO 01/21596 describesquinazoline derivatives to inhibit aurora-2 kinase. More than 30 smallmolecule Aurora kinase inhibitors are in different stages of preclinicaland clinical development (Lapenna & Giordano Nature Reviews DrugDiscovery 8: 547-566, 2009, and supplementary information; Kollareddy etal., Invest New Drugs 30(6): 2411-2432, 2012).

A cell cycle inhibitor, JNJ-7706621, shows potent inhibition of severalcyclin-dependent kinases (CDKs) and Aurora kinases, and selectivelyblocks proliferation of tumor cells of various origins. At lowconcentrations, JNJ-7706621 slows the growth of cells and at highconcentrations induces cytotoxicity. JNJ-7706621 treatment of cells hasshown a delayed progression through G1 of the cell cycle and an arrestof the cell cycle at the G2-M phase (Emanuel et al., Cancer Res. 65:9038-9046, 2005).

Inhibitors of CDKs are described in, for example, EP1244668, EP1507780,EP153976, EP1590341 EP1615926, WO 03/63764, U.S. Pat. Nos. 6,107,305,6,413,974, WO 1999/02162, WO 2000/12486, WO 2000/39101, WO 2001/14375,WO 2002/10162, WO 2002/04429, WO 2002/096888, and WO 2003/7076437. Anumber of adenosine δ′-triphosphate (ATP) competitive small organicmolecules as well as peptides have been reported in the literature asCDK inhibitors for the potential treatment of cancers.

Small molecular cyclin dependent kinase inhibitors are also describedin: Glab et al., FEBS Lett. 353: 207-211, 1994; Kitagawa et al.,Oncogene 8: 2425-2432, 1993; Losiewicz et al., Biochem. Biophys. Res.Commun. 201: 589-595, 1994; Carlson et al., Cancer Res. 56: 2973-2978,1996; Kelland, Expert Opin. Invest. Drugs 9: 2903-2911, 2000;Senderowicz, Invest. New Drugs 17: 313-320, 1999; and Vassilev et al.,PNAS 103(28): 10660-10665, 2006. In particular embodiments, CDKinhibitors can include: flavopiridol (Senderowicz Invest New Drugs17(3): 313-320, 1999); olomoucine (Vesely et al., Eur. J. Biochem. 224:771-786, 1994); roscovitine (Meijer et al., Eur. J. Biochem. 243:527-536, 1997); CDKi-277 (Amgen, Thousand Oaks, Calif.; Payton et al.,Cancer Res. 66: 4299-4308, 2006); RO-3306 (Vassilev et al., PNAS103(28): 10660-10665, 2006); purvalanol A (Villerbu et al., Int. J.Cancer 97: 761-769, 2002); NU6140 (Pennati et al., Mol. Cancer Ther. 4:1328-1337, 2005); s-CR8 (Bettayeb et al., Oncogene 27: 5797-5807, 2008);N-&-N1 (GP0210; Greenpharma S. A. S., Orleans, France; Bettayeb et al.,Mol. Cancer Ther. 7: 2713-2724, 2008); AZ703 (AstraZeneca, Cambridge,UK; Byth et al., Mol. Cancer Ther. 5: 655-664, 2006); JNJ-7706621(Johnson & Johnson, New Brunswick, N.J.; Emanuel et al., Cancer Res.65:9038-9046, 2005); RGB-286199 (GPC Biotech AG, Planegg, Germany; Wanget al., Proc. Amer. Assoc. Cancer Res. 46, Abstr. 4428, 2005); andSNS-032 (Sunesis Pharmaceuticals, San Francisco, Calif.; Choong et al.,Bioorg. Med. Chem. Lett. 18: 5763-5765, 2008; Fan et al., Bioorg. Med.Chem. Lett. 18: 6236-6239, 2008).

Polo-like kinase inhibitors include: Scytonemin (Stevenson et al.,Inflamm Res 51: 112-114, 2002); Wortmannin (Liu et al., Chem Biol 12:99-107, 2005); ON-01910 (or ON 01910.Na; multitargeted intravenous cellcycle inhibitor; Onconova Therapeutics Inc., Newtown, Pa.; Gumireddy etal., Cancer Cell 7: 275-286, 2005); BI-2536 (an ATP-competitiveinhibitor of PLK1; Boehringer Ingelheim, Ingelheim, Germany; Steegmaieret al., Current Biology 17: 316-322, 2007); BI 6727 (dihydropteridinonederivative inhibitor of PLK; Boehringer Ingelheim, Ingelheim, Germany;Rudolph et al., Clin Cancer Res 15(9): 3094-3102, 2009); GSK-61364 (orGSK-461364A; selective intravenous thiophene amide inhibitor of PLK1;Laquerre et al. A potent and selective Polo-like kinase 1 (Plk1)inhibitor (GSK461364) induces cell cycle arrest and growth inhibition ofcancer cell. Presented at the 98th American Association for CancerResearch Annual Meeting, Los Angeles, Calif., Apr. 14-18, 2007); HMN-214(oral stilbene derivative inhibitor of PLK1; prodrug of the active agentHMN-176; Nippon Shinyaku Co. Ltd, Kyoto, Japan; Garland et al., Clin CanRes 12:5182-5189, 2006); ZK-thiazolidinone (TAL; ATP-competitiveinhibitor of PLK1; Bayer Schering Pharma AG, Berlin, Germany; Santamariaet al., Mol Biol Cell 18:4024-4036, 2007); NMS-1 (an orally availableselective PLK1 inhibitor; Nerviano Medical Sciences, Milano, Italy;Beria et al. Antitumoral activity of pyrazoloquinazoline derivatives aspotent oral Plk-1 specific inhibitors. Presented at the 20th EuropeanOrganization for Research and Treatment of Cancer-National CancerInstitute-American Association for Cancer Research Symposium onMolecular Targets and Cancer Therapeutics, Geneva, Switzerland, Oct.21-24, 2008); CYC-800 (a benzthiazole-3-oxide derivative selective PLK1inhibitor; Cyclacel Ltd., Cambridge, UK; McInnes et al., Curr Top MedChem 5:181-197, 2005); DAP-81 (a diaminopyrimidine derivative thattargets PLKs; Rockefeller University, New York; Peters et al., Nat ChemBiol 2: 618-626, 2006); LC-445 (a specific non-ATP competitiveallosteric inhibitor of PLK3; Avalon Pharmaceuticals, Germantown, Md.;Horrigan et al. A small molecule allosteric inhibitor of Polo-likekinase 3 induces apoptosis and disrupts the integrity of the mitoticspindle apparatus in cancer cells. Presented at the 20th EuropeanOrganization for Research and Treatment of Cancer-National CancerInstitute-American Association for Cancer Research Symposium onMolecular Targets and Cancer Therapeutics, Geneva, Switzerland, Oct.21-24, 2008); centrinone (LCR-263) and centrinone-B (LCR-323)(inhibitors of PLK4; Wong et al., Science 348(6239): 1155-1160, 2015).Plk inhibitors are described in Schoffski The Oncologist 14: 559-570,2009.

Inhibitors of MPS1 kinase include NMS-P715 (a pyrazolo-quinazoline;Colombo et al., Cancer Res 70(24): 10255-10264, 2010); Mps-1-IN-1 andMps1-IN-2 (Kwiatkowski et al., Nat Chem Biol 6(5): 359-368 2010;Mps-1-IN-3 (Bakhos et al., JNCI: Journal of the National CancerInstitute 105(17): 1322-1331, 2013); and MPI-0479605 (Tardif et al., MolCancer Ther 10(12): 2267-2275, 2011.

In particular embodiments, a mitotic kinase inhibitor includesaminopyrazine inhibitors of Nek2 (Whelligan et al., J Med Chem53:7682-7698, 2010).

Inhibitors of Wee1 kinase include PD0166285 (pyrido-pyrimidinederivative that is a nonselective inhibitor of WEE1); PD0407824(pyrrolo-carbazole derivative that is a more selective inhibitor ofWEE1); WEE1 inhibitor II (pyrrolo-carbazole derivative); and4-(2-phenyl)-9-hydroxypyrrolo[3,4-c]-carbazole-1,3-(2H,6H)-dione (PHCD).De Witt Hamer et al. (2011) Clin Cancer Res; 17(13): 4200-4207; Palmeret al. (2006) J Med Chem 49: 4896-4911.

The terms “inhibitor of [a target protein]” or “[a target protein]inhibitor” are used to signify a compound that is capable of interactingwith the target protein and inhibiting its activity, such as anenzymatic activity. By way of example, inhibiting a target kinaseenzymatic activity means reducing the ability of that target kinase tophosphorylate a substrate peptide or protein. In various embodiments,such reduction of kinase activity is at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or at least about 90%. In variousembodiments, the concentration of kinase inhibitor (or anotherinhibitor) required to reduce kinase enzymatic activity of a targetkinase (or the activity of another target) is less than about 1 μM, lessthan about 500 nM, less than about 100 nM, or less than about 50 nM. Inembodiments, the concentration that is required to inhibit the enzymaticactivity of a target (such as a target kinase) is lower than theconcentration of the inhibitor that is required to inhibit the enzymaticactivity of other kinase(s), or other proteins in the same family orsharing an activity. In various embodiments, the concentration of aninhibitor that is required to reduce the enzymatic activity of a targetprotein is at least about 2-fold, at least about 5-fold, at least about10-fold, at least about 20-fold, at least about 50-fold, at least about100-fold, at least about 500-fold, or at least about 1000-fold lowerthan the concentration of the inhibitor that is required to reduceenzymatic activity of other proteins, particularly other similarproteins (such as other kinases). Inhibitors can also induce thereduction of the target proteins or the mRNA encoding the target proteinusing oligonucleotides (e.g., siRNA, antisense) by at least about 20%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, or at least about 90%of the original mRNA and/or protein level.

In particular embodiments, inhibition of a mitotic kinase, such as PLK1,can modulate the immune suppressive tumor microenvironment via reductionof, for example, phosphorylated STAT3, or other immune suppressivepathway, thereby benefiting antitumor immune response.

(III) IMMUNE CHECKPOINT INHIBITORS

Checkpoint inhibitor therapy is a recently developing form of cancerimmunotherapy. The therapy targets immune checkpoints, key regulators ofthe immune system that stimulate or inhibit its actions, which tumorscan use to protect from immune system attacks. Checkpoint therapy canblock inhibitory checkpoints, restoring immune system function (Pardoll,Nature Revs. Cancer 12(4):252-264, 2012). The first anti-cancer drugtargeting an immune checkpoint was ipilimumab, a CTLA-4 blocker approvedin the United States in 2011 (Cameron et al., Drugs 71(8):1093-1104,2011). See also Wieder et al., J Allergy Clin Immunol. 142(5):1403-1414, 2018.

Immune checkpoint inhibitors indirectly treat cancer by treating theimmune system. Inhibitors of immune checkpoints inhibit the normalimmunosuppressive function of immune checkpoint molecules, for example,by down regulation of expression of the checkpoint molecules or bybinding thereto and blocking normal receptor/ligand interactions. As theimmune checkpoint molecules put brakes on an immune system response toan antigen, so an inhibitor of an immune checkpoint molecule reducesthis immunosuppressive effect and enhances the immune response.Molecules that play a role in immune checkpoints include cytotoxicT-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 T cellreceptor (PD-1).

CTLA-4, PD-1, and their ligands are members of the CD28-B7 family ofco-signaling molecules that play important roles throughout all stagesof T-cell and other cell functions. The PD-1 receptor is expressed onthe surface of activated T cells (and B cells) and, under normalcircumstances, binds to its ligands (PD-L1 and PD-L2) that are expressedon the surface of antigen-presenting cells, such as dendritic cells ormacrophages. This interaction sends a signal into the T cell andessentially switches the T cell off or inhibits the T cell. Cancer cellstake advantage of this system by driving high levels of expression ofPD-L1 on their surface. This allows cancer cells to gain control of thePD-1 pathway and switch off T cells expressing PD-1 that may enter thetumor microenvironment, thus suppressing the anticancer immune response.The immunotherapy ipilimumab, a monoclonal antibody that targets CTLA-4on the surface of T cells, has been approved for the treatment ofmelanoma. Various new targeted immunotherapies aimed at the programmeddeath-1 (PD-1) T-cell receptor or its ligands (PD-L1 or PD-L2) may alsoprove to be effective. Additional immune checkpoint targets may alsoprove to be effective, such as T-cell Immunoglobulin domain and Mucindomain 3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3), various B7ligands, BTLA, adenosine A2A receptor (A2AR), and others.

Currently approved immune checkpoint inhibitors target include CTLA-4,PD-1, and PD-L1. PD-1 is the transmembrane programmed cell death 1protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1ligand 1, or CD274). PD-L1 on the cell surface binds to PD-1 on animmune cell surface, which inhibits immune cell activity. A key PD-L1function is regulation of T cell activities (Butte et al., Immunity27(11); 111-122, 2007; Karwacz et al., EMBO Mol. Med. 3(10:581-592,2011). It appears that cancer-mediated upregulation of PD-L1 on the cellsurface may inhibit T cells that might otherwise attack cancer cells.Antibodies that bind to either PD-1 or PD-L1 and therefore block theinteraction may allow the T-cells to attack the tumor (Syn et al., TheLancet Oncology 18(12):e731-e741, 2017).

In the immune system, the critical balance between rejection andself-tolerance is maintained by a finely tuned series of co-regulatoryreceptor-ligand interactions. Recent attention has focused on theprogrammed death (PD)-1/PD-1 ligand (PD-L1, B7-H1) pathway as a keymediator of tumor immune tolerance. Under physiologic conditions, theinhibitory PD-1 receptor is expressed on activated immune effectorcells, including T, B and NK cells. Through interactions with itsligands PD-L1 and PD-L2, normally expressed on antigen presenting cells(APCs), immune effector activity in peripheral tissues duringinflammatory processes is self-limited. This inhibitory system isfundamental to protecting healthy tissues and non-infected cells duringclearance of viral and bacterial intracellular infections. However, manyhuman cancers have been shown to express PD-1 ligands, thus inducingimmune tolerance locally in the tumor microenvironment (TME) andfacilitating tumor cell escape from immune attack. Two generalmechanisms promoting expression of PD-L1 on tumor cells have beenpostulated. In some tumors, aberrant signaling pathways canconstitutively up-regulate PD-L1 expression, a phenomenon termed “innateimmune resistance”; in others, the expression of PD-L1 is an adaptivemechanism that occurs in response to inflammatory cytokines produced inthe TME during an antitumor immune response (“adaptive immuneresistance”). These mechanisms of PD-L1 expression are not mutuallyexclusive, i.e., constitutive PD-L1 expression on tumor cells may befurther up-regulated by cytokines such as interferon-gamma (IFN-g).

PD-L1 expression by tumor cells prior to treatment correlates highlywith response to anti-PD-1 monotherapy (for example, nivolumab(Bristol-Myers Squibb; OPDIVO™), pembrolizumab (Merck; KEYTRUDA®)) andanti-PD-L1 therapy (for example, MPDL3280A (Genentech/Roche)).Additional checkpoint inhibitors include: ipilimumab and tremelimumab(which target CTLA-4); atezolizumab (Genentech/Roche; Tecentriq),avelumab (Merck; Bavencio), and durvalumab (Medimmune/Strazeneca;Imfinzi) (which target PD-L1); and cemiplimab (REGN-2810), nivolumab,pembrolizumab, and pidilizumab (which target PD-1). Spartalizumab(PDR001; Novartis) is also under development as a PD-1 inhibitor.

Methods of PD-1 blockade treatment, including treatment of cancers, arewell known in the art. See, for instance, WO 2016/201425, US2019/0275705, Kvistborg et al. (Science Transl Med. 6(254):254ra128,2014), Zou et al. (Science Transl Med. 8(328):328rv4, 2016), andSakuishi et al. J Exp Med. 207(10):2187-2194, 2010).

PD-1 blocking agents include those used to treat cancer (i.e., toinhibit the growth or survival of tumor cells). Cancers whose growth maybe inhibited using antibodies or anti-PD-1 agents or other check pointinhibitors include cancers typically responsive to immunotherapy, butalso cancers that have not hitherto been associated with immunotherapy.Examples of cancers for treatment include melanoma (e.g., metastaticmalignant melanoma), renal cancer (e.g., clear cell carcinoma), prostatecancer (e.g., hormone refractory prostate adenocarcinoma), pancreaticadenocarcinoma, breast cancer, colon cancer, lung cancer (e.g.,non-small cell lung cancer), esophageal cancer, squamous cell carcinomaof the head and neck, liver cancer, ovarian cancer, cervical cancer,thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and otherneoplastic malignancies. The herein described treatments are applicableto malignancies that demonstrate improved disease-free and overallsurvival in relation to the presence of tumor-infiltrating lymphocytesin biopsy or surgical material, e.g., melanoma, colorectal, liver,kidney, stomach/esophageal, breast, pancreas, and ovarian cancer. Suchcancer subtypes are known to be susceptible to immune control by Tlymphocytes. Additionally, the provided technology is useful fortreating refractory or recurrent malignancies whose growth may beinhibited using the PD-1 or other check point blockade treatments.Particularly cancers include those characterized by elevated expressionof PD-1 and/or its ligands PD-L1 and/or PD-L2 in tested tissue samples,including: ovarian, renal, colorectal, pancreatic, breast, liver,glioblastoma, non-small cell lung cancer, gastric, esophageal cancersand melanoma. Cancers also include those associated with persistentinfection with viruses such as human immunodeficiency viruses, hepatitisviruses class A, B and C, Epstein Barr virus, human papilloma virusesthat are known to be causally related to for instance Kaposi's sarcoma,liver cancer, nasopharyngeal cancer, lymphoma, cervical, vulval, anal,penile, and oral cancers.

The PD-1/PD-L1 pathway is a well-validated target for the development ofantibody therapeutics for cancer treatment. Anti-PD-1 antibodies mayalso be useful in chronic viral infection. Memory CD8+ T cells generatedafter an acute viral infection are highly functional and constitute animportant component of protective immunity. In contrast, chronicinfections are often characterized by varying degrees of functionalimpairment (exhaustion) of virus-specific T-cell responses, and thisdefect is a principal reason for the inability of the host to eliminatethe persisting pathogen. Although functional effector T cells areinitially generated during the early stages of infection, they graduallylose function during the course of a chronic infection. Barber et al.(Nature 439: 682-687, 2006) showed that mice infected with a laboratorystrain of LCMV developed chronic infection resulting in high levels ofvirus in the blood and other tissues. These mice initially developed arobust T cell response, but eventually succumbed to the infection upon Tcell exhaustion. The authors found that the decline in number andfunction of the effector T cells in chronically infected mice could bereversed by injecting an antibody that blocked the interaction betweenPD-1 and PD-L1.

In particular embodiments, immune checkpoint molecules include CTLA-4,PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Killer-cell Immunoglobulin-likeReceptor (KIR), CD160, B7-H3 (CD276), BTLA (CD272), IDO (Indoleamine2,3-dioxygenase), adenosine A2A receptor (A2AR), and C100RF54.

The term “immune checkpoint protein” or “immune checkpoint molecule”refers to a molecule that is expressed by T cells and that either turnup a signal (stimulatory checkpoint molecules) or turn down a signal(inhibitory checkpoint molecules). Immune checkpoint molecules arerecognized in the art to constitute immune checkpoint pathways similarto the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, Nature RevCancer 12:252-264, 2012; Mellman et al., Nature 480: 480-489, 2011).Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4,BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. TheAdenosine A2A receptor (A2AR) is regarded as an important checkpoint incancer therapy because the tumor microenvironment has relatively highlevels of adenosine, which lead to a negative immune feedback loopthrough the activation of A2AR. B7-H3, also called CD276, was originallyunderstood to be a co-stimulatory molecule but is now regarded asco-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells andtumor-associated macrophages and plays a role in tumor escape. B and TLymphocyte Attenuator (BTLA), also called CD272, is a ligand of HVEM(Herpesvirus Entry Mediator). Cell surface expression of BTLA isgradually downregulated during differentiation of human CD8+ T cellsfrom the naive to effector cell phenotype; however, tumor-specific humanCD8+ T cells express high levels of BTLA. CTLA-4, also called CD152, isoverexpressed on regulatory T (Treg) cells and serves to control T cellproliferation. IDO is a tryptophan catabolic enzyme in the tryptophan tokynurenine metabolic pathway that regulates innate and adaptiveimmunity. IDO is known to suppress T and natural killer (NK) cells,generate and activate Tregs and myeloid-derived suppressor cells, andpromote tumor angiogenesis. Another important molecule is TDO,tryptophan 2,3-dioxygenase, a key enzyme in the tryptophan to kynureninemetabolic pathway (Platten et al., Front Immunol. 5: 673, 2014). KIR isa receptor for MHC Class I molecules on NK cells. LAG-3 works tosuppress an immune response by action on Tregs as well as direct effectson CD8+ T cells. PD-1, Programmed Death 1 (PD-1) receptor, has twoligands, PD-L1 and PD-L2. This checkpoint is the target of melanoma drugKeytruda® (pembrolizumab, Merck & Co., Kenilworth, N.J.), which gainedFDA approval in September 2014. An advantage of targeting PD-1 is thatit can restore immune function in the tumor microenvironment. TIM-3 isexpressed on activated human CD4+ T cells and regulates Th1 and Th17cytokines. TIM-3 acts as a negative regulator of Th1/Tel function bytriggering cell death upon interaction with its ligand, galectin-9.V-domain Ig suppressor of T cell activation (VISTA) is primarilyexpressed on hematopoietic cells so that consistent expression of VISTAon leukocytes within tumors may allow VISTA blockade to be effectiveacross a broad range of solid tumors.

The term “immune checkpoint inhibitor” refers to any compound inhibitingthe function of an immune inhibitory checkpoint protein. Inhibitionincludes reduction of function and full blockade. In particularembodiments, immune checkpoint inhibitors are antibodies thatspecifically recognize an immune checkpoint protein. In particularembodiments, immune checkpoint inhibitors include peptides, antibodies,nucleic acid molecules, and small molecules. In particular embodiments,an immune checkpoint inhibitor is administered for enhancing theproliferation, migration, persistence and/or cytotoxic activity of CD8+T cells in the subject and in particular the tumor-infiltrating CD8+ Tcells of the subject.

Immune checkpoint inhibitors include agents that inhibit (directly orindirectly) at least one of CTLA-4, PD-1, PD-L1, and the like. Suitableanti-CTLA-4 therapy agents for use in the methods of the disclosureinclude anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouseanti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanizedanti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonalanti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, ipilimumab,tremelimumab, anti-CD28 antibodies, anti-CTLA-4 ADNECTINS™, anti-CTLA-4domain antibodies, single chain anti-CTLA-4 fragments, heavy chainanti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors ofCTLA-4 that agonize the co-stimulatory pathway, the antibodies disclosedin WO 2001/014424, the antibodies disclosed in WO 2004/035607, theantibodies disclosed in US 2005/0201994, and the antibodies disclosed inEP1212422B1. Additional anti-CTLA-4 antibodies are described in U.S.Pat. Nos. 5,811,097; 5,855,887; 6,051,227; 6,984,720; WO 01/14424; WO00/37504; US 2002/0039581; and US 2002/086014. Other anti-CTLA-4antibodies that can be used in a method of the present disclosureinclude, for example, those disclosed in: WO 98/42752; U.S. Pat. Nos.6,682,736; 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA,95(17): 10067-10071, 1998; Camacho et al., J. Clin. Oncology, 22(145):Abstract No. 2505, 2004 (antibody CP-675206); Mokyr et al., Cancer Res58: 5301-5304, 1998; U.S. Pat. No. 5,977,318: U.S. Pat. Nos. 6,682,736;7,109,003; and 7,132,281.

Suitable anti-PD-1 and anti-PD-L1 therapy agents for use in the methodsof the disclosure include anti-PD-1 and anti-PD-L1 antibodies, humananti-PD-1 and anti-PD-L1 antibodies, mouse anti-PD-1 and anti-PD-L1antibodies, mammalian anti-PD-1 and anti-PD-L1 antibodies, humanizedanti-PD-1 and anti-PD-L1 antibodies, monoclonal anti-PD-1 and anti-PD-L1antibodies, polyclonal anti-PD-1 and anti-PD-L1 antibodies, chimericanti-PD-1 and anti-PD-L1 antibodies. In particular embodiments,anti-PD-1 therapy agents include nivolumab, pembrolizumab, pidilizumab,MED10680 (AstraZeneca, Cambridge, UK), and combinations thereof. Inparticular embodiments, anti-PD-L1 therapy agents include atezolizumab,BMS-936559 (Bristol-Myers Squibb, New York, N.Y.), durvalumab(MED14736), avelumab (MSB0010718C), and combinations thereof.

Suitable anti-PD-1 and anti-PD-L1 antibodies are described in Topalianet al. (Cancer Cell 27: 450-461, 2015).

In particular embodiments, immune checkpoint inhibitors can include amodified ligand or an antisense nucleic acid molecule such as siRNAdesigned to inhibit a particular immune checkpoint molecule. Inparticular embodiments, the siRNA prevents the translation of the immunecheckpoint molecule, thus preventing the expression of the protein.Given that the genomic sequences of many immune checkpoint molecules areknown, one of ordinary skill in the art would be able to use routinemethods to design suitable inhibitory antisense nucleic acid molecules.

In certain embodiments, checkpoint inhibitors can be siRNA, smallmolecule inhibitors, or antibody against (specific for) an immunecheckpoint molecule beneficial for cancer treatment. Such targetsinclude PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, B7-H3, VISTA, A2AR, and IDO(Khair et al., Frontiers Immunology, 10:453, 2019).

One of ordinary skill in the art will understand how to accessrepresentative sequences for such targets, which are readily availablein public sequence databases. The following table provides samplesequence information:

Gene Abbreviation Full gene name Representative GenBank Accession #sPD-L1 CD274 molecule NM_001267706.1; NM_001314029.1; NM_014143.3;NR_052005.1 PD-1 programmed cell death 1 NM_005018.2; XM_006712573.2;XM_017004293.1 CTLA-4 cytotoxic T-lymphocyte NM_001037631.2; NM_005214.4associated protein 4 LAG3 Lymphocyte activating 3 NM_002286.5;XM_011520956.1 TIM-3 T-cell immunoglobulin and NM_032782.4 mucin-domaincontaining-3 B7-H3 CD276 (Cluster of NM_001024736.1; NM_001329628.1;Differentiation 276) VISTA V-domain Ig suppressor of T NM_001329629.1;NM_025240.2; cell activation A2AR adenosine A2a receptor XM_005254700.4;XM_011522095.2; IDO indoleamine 2,3-dioxygenase XM_011522096.2;XM_017022638.1

(IV) OPTIONAL ADDITIONAL COMPONENTS

In addition to the mitotic kinase inhibitor and the immune checkpointinhibitor, the therapeutic constructs provided herein can optionallycontain or be administered with one or more optional components. Theseoptional components include adjuvant(s), therapeutic oligonucleotides,additional anti-cancer agent(s), and targeting moieties.

Adjuvants

The therapeutic constructs provided herein optionally may include atleast one adjuvant component, contained within or otherwise associatedwith the delivery vehicle. The therapeutic construct embodiments are notlimited to a particular type of adjuvant, though specific examples areprovided herein.

Generally, adjuvants are any substance whose admixture into a vaccinecomposition increases or otherwise modifies the immune response to the(cancer) antigen. The ability of an adjuvant to increase the immuneresponse to an antigen is typically manifested by a significant increasein immune-mediated reaction, or reduction in disease symptoms. Forexample, an increase in humoral immunity is typically manifested by asignificant increase in the titer of antibodies raised to the antigen,and an increase in T-cell activity is typically manifested in increasedantigen-specific T cell proliferation, death of target cells, orcytokine secretion. An adjuvant may also alter an immune response, forexample, by changing a primarily humoral or Th2 response into aprimarily cellular, or Th1 response.

Suitable adjuvants include, but are not limited to TLR-binding DNAsubstituents such as CpG oligonucleotides (e.g., ISS 1018; Amplivax; CpGODN 7909, CpG ODN 1826, CpG ODN D19, CpG ODN 1585, CpG ODN 2216, CpG ODN2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN 2395, ODN M362, andSD-101), DNA TLR agonists that contain a CpG sequence (e.g., dSLIM),non-CpG DNA TLR agonists (e.g., EnanDIM), and cationicpeptide-conjugated CpG oligonucleotides (e.g., IC30, IC31); RNA TLRagonists (e.g., Poly I:C and Poly-ICLC); aluminum salts (e.g., aluminumhydroxide, aluminum phosphate, aluminum chloride, and aluminum potassiumsulfate); anti-CD40 antibodies (e.g., CP-870,893); cytokines, such asgranulocyte-macrophage colony-stimulating factor (GM-CSF); smallmolecule TLR agonists (e.g., imiquimod, resiquimod, gardiquimod, and3M-052); fusion proteins (e.g., ImuFact IMP321, CyaA, and ONTAK);oil-orsurfactant-based adjuvants such as MF59, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, and Montanide ISA-51; a plantextract such as QS21 stimulon (Aquila Biotech, Worcester, Mass., USA),which is derived from saponin; mycobacterial extracts and syntheticbacterial cell wall mimics, such as lipopolysaccharides (e.g.,monophosphoryl lipid A, OM-174, OM-197-MP-EC, and Pam3Cys); xanthenonederivatives (e.g., vadimezan); mixtures thereof (e.g., AS-15); and otherproprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Severalimmunological adjuvants (e.g., MF59 specific for dendritic cells andtheir preparation have been described previously (Dupuis et al., CellImmunol. 186(1): 18-27, 1998; Allison, Dev Biol Stand.; 92:3-11, 1998).Also cytokines may be used as adjuvants. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-alpha), accelerating the maturation of dendriticcells into efficient antigen-presenting cells for T-lymphocytes (e.g.,GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589) and acting asimmunoadjuvants (e.g., IL-12) (Gabrilovich et al., J Immunother EmphasisTumor Immunol. (6):414-418, 1996). Toll like receptors (TLRs) or agentsthat activate TLRs may also be used as adjuvants, and are importantmembers of the family of pattern recognition receptors (PRRs) whichrecognize conserved motifs shared by many micro-organisms, termed“pathogen-associated molecular patterns” (PAMPS).

In some embodiments, the adjuvant includes a CpG oligonucleotide. CpGimmuno-stimulatory oligonucleotides have also been reported to enhancethe effects of adjuvants in a vaccine setting. Without being bound byany particularly mechanistic theory, CpG oligonucleotides act at leastin part by activating the innate (non-adaptive) immune system viaToll-like receptors (TLR), mainly TLR9. CpG triggered TLR9 activationenhances antigen-specific humoral and cellular responses to a widevariety of antigens, including peptide or protein antigens, live orkilled viruses, dendritic cell vaccines, autologous cellular vaccinesand polysaccharide conjugates in both prophylactic and therapeuticvaccines. More importantly, it enhances dendritic cell maturation anddifferentiation, resulting in enhanced activation of T_(H)1 cells andstrong cytotoxic T-lymphocyte (CTL) generation, even in the absence ofCD4 T-cell help. The T_(H)1 bias induced by TLR9 stimulation ismaintained even in the presence of vaccine adjuvants such as alum orincomplete Freund's adjuvant (IFA) that normally promote a T_(H)2 bias.CpG oligonucleotides show even greater adjuvant activity when formulatedor co-administered with other adjuvants or in formulations such asmicroparticles, nano particles, lipid emulsions or similar formulations,which are especially necessary for inducing a strong response when theantigen is relatively weak. They also accelerate the immune response andenabled the antigen doses to be reduced by approximately two orders ofmagnitude, with comparable antibody responses to the full-dose vaccinewithout CpG in some experiments (Krieg, Nature Reviews, Drug Discovery,5:471-484, 2006). U.S. Pat. No. 6,406,705 describes the combined use ofCpG oligonucleotides, non-nucleic acid adjuvants and an antigen toinduce an antigen-specific immune response. A commercially available CpGTLR9 agonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, GERMANY). Other TLR binding molecules such as RNA binding TLR7, TLR 8 and/or TLR 9 may also be used.

Xanthenone derivatives such as, for example, vadimezan or AsA404 (alsoknown as 5,6-dimethylaxanthenone-4-acetic acid (DMXAA)), may also beused as adjuvants according to embodiments of the invention.Alternatively, such derivatives may also be administered in parallel tothe vaccine of the invention, for example via systemic or intratumoraldelivery, to stimulate immunity at the tumor site. Without being boundby theory, it is believed that such xanthenone derivatives act bystimulating interferon (IFN) production via the stimulator of IFN gene(STING) receptor (see e.g., Conlon et al., J Immunology, 190:5216-5225,2013; and Kim et al., ACS Chem Biol, 8:1396-1401, 2013). Other examplesof useful adjuvants include, but are not limited to, chemically modifiedCpGs (e.g. CpR, Idera), Poly(I:C) (e.g. polyi:Cl2U), non-CpG bacterialDNA or RNA as well as immunoactive small molecules and antibodies suchas cyclophosphamide, sunitinib, bevacizumab, Celebrex™, NCX-4016,sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632,pazopanib, AZD2171, ipilimumab, tremelimumab, and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation. Additional adjuvants include colony-stimulatingfactors, such as Granulocyte Macrophage Colony Stimulating Factor(GM-CSF, sargramostim).

Poly-ICLC is a synthetically prepared double-stranded RNA consisting ofpolyI and polyC strands of average length of about 5000 nucleotides,which has been stabilized to thermal denaturation and hydrolysis byserum nucleases by the addition of poly-lysine andcarboxymethylcellulose. The compound activates TLR3 and the RNAhelicase-domain of MDA5, both members of the PAMP family, leading to DCand natural killer (NK) cell activation and production of a “naturalmix” of type I interferons, cytokines, and chemokines. Furthermore,poly-ICLC exerts a more direct, broad host-targeted anti-infectious andpossibly antitumor effect mediated by the two IFN-inducible nuclearenzyme systems, the 2′ 5′-OAS and the PI/elF2a kinase, also known as thePKR (4-6), as well as RIG-1 helicase and MDA5.

Examples of immunological adjuvants that can be associated with thetherapeutic constructs include TLR ligands, C-Type Lectin Receptorligands, NOD-Like Receptor ligands, RLR ligands, and RAGE ligands. TLRligands can include lipopolysaccharide (LPS) and derivatives thereof, aswell as lipid A and derivatives thereof including, but not limited to,monophosphoryl lipid A (MPL), glycopyranosyl lipid A, PET-lipid A, and3-O-desacyl-4′-monophosphoryl lipid A. In a specific embodiment, theimmunological adjuvant is MPL. In another embodiment, the immunologicaladjuvant is LPS. TLR ligands can also include, but are not limited to,TLR3 ligands (e.g., polyinosinic-polycytidylic acid (poly(I:C)), TLR7ligands (e.g., imiquimod and resiquimod), and TLR9 ligands.

As used herein, the term “TLR-binding DNA substituent” refers to asubstituent or moiety capable of binding to a toll-like receptor(“TLR”), including at least one deoxyribonucleic acid. In embodiments, aTLR-binding DNA substituent is a nucleic acid. In embodiments, theTLR-binding DNA substituent includes at least one nucleic acid analog.In embodiments, the TLR-binding DNA substituent includes at least onenucleic acid analog having an alternate backbone (e.g. phosphodiesterderivative (e.g. phosphoramidate, phosphorodiamidate, phosphorothioate,phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates,phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boronphosphonate, or O-methylphosphoroamidite), peptide nucleic acidbackbone(s), LNA, or linkages). In embodiments, a TLR-binding DNAsubstituent includes DNA. In embodiments, all nucleotide sugars in aTLR-binding DNA substituent are deoxyribose (e.g., all nucleotides areDNA). In embodiments, a TLR-binding DNA substituent consists of DNA. Inembodiments, a TLR-binding DNA substituent includes or is DNA havinginternucleotide linkages selected from phosphodiesters andphosphodiester derivatives (e.g. phosphoramidate, phosphorodiamidate,phosphorothioate, phosphorodithioate, phosphonocarboxylic acids,phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,methyl phosphonate, boron phosphonate, O-methylphosphoroamidite, orcombinations thereof). In embodiments, a TLR-binding DNA substituentconsists of DNA having internucleotide linkages selected fromphosphodiesters and phosphorothioates. In embodiments, a TLR-binding DNAsubstituent includes or is DNA having backbone linkages selected fromphosphodiesters and phosphorodithioates. In embodiments, a TLR-bindingDNA substituent includes or is DNA including phosphodiester backbonelinkages. In embodiments, a TLR-binding DNA substituent includes or isDNA including phosphorothioate backbone linkages. In embodiments, aTLR-binding DNA substituent includes or is DNA includingphosphorodithioate backbone linkages. In embodiments, a TLR-binding DNAsubstituent preferentially binds TLR9 over other TLR. In embodiments, aTLR-binding DNA substituent specifically binds TLR9. In embodiments, aTLR-binding DNA substituent specifically binds TLR3. In embodiments, aTLR-binding DNA substituent specifically binds TLR7. In embodiments, aTLR-binding DNA substituent specifically binds TLR8. In embodiments, aTLR-binding DNA substituent specifically binds a cellularsub-compartment (e.g. endosome) associated TLR (e.g. TLR3, TLR7, TLR8,or TLR9). In embodiments, a TLR-binding DNA substituent includes or is aG-rich oligonucleotide. In embodiments, a TLR-binding DNA substituentincludes a CpG motif, wherein C and G are nucleotides and p is thephosphate connecting the C and G. In embodiments, the CpG motif isunmethylated. In embodiments, a TLR-binding DNA substituent is a Class ACpG oligodeoxynucleotide (ODN). In embodiments, a TLR-binding DNAsubstituent is a Class B CpG oligodeoxynucleotide (ODN). In embodiments,a TLR-binding DNA substituent is a Class C CpG oligodeoxynucleotide(ODN). In embodiments, a TLR-binding DNA substituent (e.g., TLR9-bindingDNA substituent) consists of deoxyribonucleic acids with A, G, C, or Tbases and phosphodiester linkages and/or phosphodiester derivativelinkages (e.g., phosphorothioate linkage(s)).

The phrase “CpG motif” refers to a 5′ C nucleotide connected to a 3′ Gnucleotide through a phosphodiester internucleotide linkage or aphosphodiester derivative internucleotide linkage. In embodiments, a CpGmotif includes a phosphodiester internucleotide linkage. In embodiments,a CpG motif includes a phosphodiester derivative internucleotidelinkage.

As used herein, the term “Class A CpG ODN” or “A-class CpG ODN” or“D-type CpG ODN” or “Class A CpG DNA sequence” is used in accordancewith its common meaning in the biological and chemical sciences andrefers to a CpG motif including oligodeoxynucleotide including one ormore of poly-G sequence at the 5′, 3′, or both ends; an internalpalindrome sequence including CpG motif; or one or more phosphodiesterderivatives linking deoxynucleotides. In embodiments, a Class A CpG ODNincludes poly-G sequence at the 5′, 3′, or both ends; an internalpalindrome sequence including CpG motif; and one or more phosphodiesterderivatives linking deoxynucleotides. In embodiments, the phosphodiesterderivative is phosphorothioate. Examples of Class A CpG ODNs include ODND19, ODN 1585, ODN 2216, and ODN 2336.

The terms “Class B CpG ODN” or “B-class CpG ODN” or “K-type CpG ODN” or“Class B CpG DNA sequence” are used in accordance with their commonmeaning in the biological and chemical sciences, and refer to a CpGmotif including oligodeoxynucleotide including one or more of a 6mermotif including a CpG motif; phosphodiester derivatives linking alldeoxynucleotides. In embodiments, a Class B CpG ODN includes one or morecopies of a 6mer motif including a CpG motif and phosphodiesterderivatives linking all deoxynucleotides. In embodiments, thephosphodiester derivative is phosphorothioate. In embodiments, a Class BCpG ODN includes one 6mer motif including a CpG motif. In embodiments, aClass B CpG ODN includes two copies of a 6mer motif including a CpGmotif. In embodiments, a Class B CpG ODN includes three copies of a 6mermotif including a CpG motif. In embodiments, a Class B CpG ODN includesfour copies of a 6mer motif including a CpG motif. Examples of Class BCpG ODNs include ODN 1668, ODN 1826, ODN 2006, and ODN 2007.

The terms “Class C CpG ODN” or “C-class CpG ODN” or “C-type CpG DNAsequence” are used in accordance with their common meaning in thebiological and chemical sciences and refers to an oligodeoxynucleotideincluding a palindrome sequence including a CpG motif and phosphodiesterderivatives (phosphorothioate) linking all deoxynucleotides. Examples ofClass C CpG ODNs include ODN 2395 and ODN M362.

Therapeutic Oligonucleotides.

Optionally, the provided therapeutic constructs may contain one or moretherapeutic oligonucleotides. Different types of therapeuticoligonucleotides can be used and non-exhaustively include siRNA, miRNA,antisense oligonucleotide, ribozyme, aptamer, DNA, mRNA, sgRNA (forCRISPR), and CRISPR-cas9 elements. In other words, any chain ofnucleotides can be utilized as long as they can specifically modulate(interfere or boost) the action or synthesis of certain gene(s) andprotein(s). Each particular oligonucleotide may have a single ormultiple targets. Examples of gene/protein targets of interest to theinvention include immune checkpoints (discussed elsewhere herein),transcription factors, phosphatases, kinases, etc. Specific targetsinclude, but are not limited to, STAT3, TGF-β, CD47, NOX1-5, HSP47,XBP1, BCL2, BCL-XL, AKT1, AKT2, AKT3, MYC, HER2, HER3, AR, Survivin,GRB7, EPS8L1, RRM2, PKN3, EGFR, IRE1-alpha, VEGF-R1, RTP801, proNGF,Keratin K6A, LMP2, LMP7, MECL1, HIF1α, Furin, KSP, eiF-4E, p53,β-catenin, ApoB, PCSK9, SNALP, CD39, CD73, MIF, VEGF, PIGF, CXCR4, CCR2,PLK1, MTDH, Twist, Lcn2, IL-6, IL-10, p65, and mitotic kinases (e.g.,PLK1, PLK2, PLK3, PLK4, CDK1, CDK2, CHK1, CHK2, BUB1, BUBR1, MPS1, NEK2,HASPIN, Aurora A) as previously mentioned. Therapeutic oligonucleotidescan also contain two strands that target two genes (such as siRNAagainst BCL2 and AKT1, siRNA against AR and MYC). They can also containimmunostimulatory sequences/elements that can thus simultaneously boostthe immune response and regulate expression of target genes. They canalso be designed to target the aforementioned genes that have mutations.

In certain embodiments, the therapeutic constructs include as an activeagent an oligonucleotide that mediates RNA interference. RNAinterference is a highly conserved mechanism triggered bydouble-stranded RNA (dsRNA) and able to downregulate transcript of geneshomologous to the dsRNA. The dsRNA is first processed by Dicer intoshort duplexes of 21-23 nucleotides, called short interfering RNAs(siRNAs). Incorporated in RNA-induced silencing complex (RISC), they areable to mediate gene silencing through cleavage of the target mRNA.“siRNA” or “small-interfering ribonucleic acid” refers to two strands ofribonucleotides which hybridize along a complementary region underphysiological conditions. The siRNA molecules comprise a double-strandedregion which is substantially identical to a region of the mRNA of thetarget gene. A region with 100% identity to the corresponding sequenceof the target gene is suitable. This state is referred to as “fullycomplementary”. However, the region may also contain one, two or threemismatches as compared to the corresponding region of the target gene,depending on the length of the region of the mRNA that is targeted, andas such may be not fully complementary. Methods to analyze and identifysiRNAs with sufficient sequence identity in order to effectively inhibitexpression of a specific target sequence are known in the art. Asuitable mRNA target region would be the coding region. Also suitableare untranslated regions, such as the 5-UTR, the 3-UTR, and splicejunctions as long as the regions are unique to the mRNA target and notdirected to a mRNA poly A tail.

In some embodiments, siRNA encapsulated within or associated withtherapeutic constructs are utilized in methods and systems involving RNAinterference. Such embodiments are not limited to a particular size ortype of siRNA molecule. The length of the region of the siRNAcomplementary to the target, for example, may be from 15 to 100nucleotides, 18 to 25 nucleotides, 20 to 23 nucleotides, or more than15, 16, 17 or 18 nucleotides. Where there are mismatches to thecorresponding target region, the length of the complementary region isgenerally required to be somewhat longer.

In certain embodiments, it is contemplated that the siRNA deliveryapproach using therapeutic constructs disclosed herein (e.g., throughloading of the siRNA on a therapeutic constructs) can be used to inhibitproduction of any gene of interest. Specific targets include, but arenot limited to, STAT3, TGF-β, CD47, NOX1-5, HSP47, XBP1, BCL2, BCL-XL,AKT1, AKT2, AKT3, MYC, HER2, HER3, AR, Survivin, GRB7, EPS8L1, RRM2,PKN3, EGFR, IRE-alpha, VEG-R, RTP81, proNGF, Keratin K6A, LMP2, LMP7,MECL1, HIF1α, Furin, KSP, eiF-4E, p53, β-catenin, ApoB, PCSK9, SNALP,CD39, CD73, MIF, VEGF, PIGF, CXCR4, CCR2, PLK1, MTDH, Twist, Lcn2, IL-6,IL-10, p65, and mitotic kinases as previously mentioned among genesknown as drivers in cancer and other diseases. Further, it isspecifically contemplated that siRNA can be directed to a variant ormutated gene, rather than a wildtype gene.

One of ordinary skill in the art will understand how to accessrepresentative sequences for these targets, which are readily availablein public sequence databases. The following table provides samplesequence information:

Gene Abbreviation Full gene name Representative GenBank Accession #sSTAT3 Signal transducer and NM_003150.3; NM_139276.2; NM_213662.1;activator of transcription 3 XM_005257616.3; XM_005257617.3;XM_011525145.2; XM_011525146.2; XM_017024972.1; XM_017024973.1;XM_017024974.1; XM_017024975.1; XM_017024976.1 TGF-β transforming growthfactor NM_000660.6; XM_011527242.1 beta 1 CD47 CD47 moleculeNM_001777.3; NM_198793.2; XM_005247908.1; XM_005247909.1;XM_017007536.1; XR_001740374.1; XR_001740375.1; XR_241521.1;XR_241522.1; XR_924218.1; XR_924219.1; XR_924220.1 NOX1 NADPH oxidase 1NM_001271815.1; NM_007052.4; NM_013955.2; XM_017029407.1 NOX2 cytochromeb-245 beta NM_000397.3 chain NOX3 NADPH oxidase 3 NM_015718.2 NOX4 NADPHoxidase 4 NM_001143836.2; NM_001143837.1; NM_001291926.1;NM_001291927.1; NM_001291929.1; NM_001300995.1; NM_016931.4;XM_006718849.3; XM_011542857.2; XM_017017841.1; XM_017017842.1;XM_017017843.1; XM_017017844.1; XM_017017845.1; NR_120406.1 NOX5 NADPHoxidase 5 NM_001184779.1; NM_001184780.1; NM_024505.3; NR_033671.2;NR_033672.1 HSP47 serpin family H member 1 NM_001207014.1; NM_001235.3;XM_011545327.1 XBP1 X-box binding protein 1 NM_001079539.1; NM_005080.3BCL2 B-cell lymphoma 2, NM_000633.2; NM_000657.2; XM_011526135.2;apoptosis regulator XM_017025917.1; XR_935248.2 BCL-XL/S, B-celllymphoma 2 like 1 NM_001191.3; NM_001317919.1; BCL2L, BCLX,NM_001317920.1; NM_001317921.1; Bcl-X, NM_001322239.1; NM_001322240.1;PPP1R52 NM_001322242.1; NM_138578.2; XM_011528964.2; XM_017027993.1;NR_134257.1; XR_001754364.1; XR_936599.2 AKT1 AKT serine/threonineNM_001014431.1; NM_001014432.1; kinase 1 NM_005163.2; XM_005267401.1;XM_017021075.1; XM_017021076.1; XM_017021077.1; XM_017021078.1 AKT2 AKTserine/threonine NM_001243027.2; NM_001243028.2; kinase 2NM_001330511.1; NM_001626.5; XM_011526614.1; XM_011526615.1;XM_011526616.1; XM_011526618.1; XM_011526619.1; XM_011526620.1;XM_011526622.2; XM_017026470.1 AKT3 AKT serine/threonine NM_001206729.1;NM_005465.4; NM_181690.2; kinase 3 XM_005272994.4; XM_005272995.2;XM_006711726.3; XM_011544012.2; XM_011544013.2; XM_011544014.2;XM_016999985.1 MYC MYC proto-oncogene, NM_002467.4 bHLH transcriptionfactor HER2 erb-b2 receptor tyrosine NM_001005862.2; NM_001289936.1;kinase 2 NM_001289937.1; NM_001289938.1; NM_004448.3; NR_110535.1 HER3erb-b2 receptor tyrosine NM_001005915.1; NM_001982.3 kinase 3 ARandrogen receptor NM_000044.4; NM_001011645.3; NM_001348061.1;NM_001348063.1; NM_001348064.1 Survivin baculoviral inhibitor ofNM_001012270.1; NM_001012271.1; (BIRC5) apoptosis repeat- NM_001168.2;XR_243654.4; XR_934452.2 containing 5 GRB7 growth factor receptorNM_001030002.2; NM_001242442.1; bound protein 7 NM_001242443.1;NM_001330207.1; NM_005310.3; XM_017024536.1; XM_017024538.1 EPS8L1 EPS8like 1 NM_017729.3; NM_133180.2; XM_005259020.1; XM_011527050.1;XM_011527051.2; XM_011527052.2 RRM2 ribonucleotide reductaseNM_001034.3; NM_001165931.1 regulatory subunit M2 PKN3 protein kinase N3NM_001317926.1; NM_013355.4; XM_005251946.3; XM_006717080.2;XM_017014649.1; XM_017014650.1 EGFR epidermal growth factorNM_001346897.1; NM_001346898.1; receptor NM_001346899.1; NM_001346900.1;NM_001346941.1; NM_005228.4; NM_201282.1; NM_201283.1; NM_201284.1IRE1-alpha endoplasmic reticulum to NM_001433.3; XM_017024347.1; (ERN1)nucleus signaling 1 XM_017024348.1 VEGF-R1 fms related tyrosineNM_001159920.1; NM_001160030.1; (FLT1) kinase 1 NM_001160031.1;NM_002019.4; XM_011535014.1; XM_017020485.1 RTP801 DNA damage inducibleNM_019058.3 (DDIT4) transcript 4 Keratin keratin 1 NM_006121.3 K6Akeratin 6A NM_005554.3 LMP2 proteasome subunit beta 9 NM_002800.4 LMP7proteasome subunit beta 8 NM_004159.4; NM_148919.3 MECL1 proteasomesubunit beta 10 NM_002801.3 HIF1α hypoxia inducible factor 1NM_001243084.1; NM_001530.3; NM_181054.2 alpha subunit Furin furin,paired basic amino NM_001289823.1; NM_001289824.1; acid cleaving enzymeNM_002569.3 KSP fibroblast growth factor NM_031950.3 binding protein 2eiF-4E eukaryotic translation NM_001130678.2; NM_001130679.2; initiationfactor 4E NM_001331017.1; NM_001968.4 p53 tumor protein p53 NM_000546.5;NM_001126112.2; NM_001126113.2; NM_001126114.2; NM_001126115.1;NM_001126116.1; NM_001126117.1; NM_001126118.1; NM_001276695.1;NM_001276696.1; NM_001276697.1; NM_001276698.1; NM_001276699.1;NM_001276760.1; NM_001276761.1 β-catenin catenin beta 1 NM_001098209.1;NM_001098210.1; NM_001330729.1; NM_001904.3; XM_005264886.2;XM_006712983.1; XM_006712984.1; XM_006712985.1; XM_017005738.1 ApoBapolipoprotein B NM_000384.2 PCSK9 proprotein convertase NM_174936.3;NR_110451.1 subtilisin/kexin type 9 SNALP synaptosome associatedNM_001322902.1; NM_001322903.1; protein 25 NM_001322904.1;NM_001322905.1; NM_001322906.1; NM_001322907.1; NM_001322908.1;NM_001322909.1; NM_001322910.1; NM_003081.4; NM_130811.3;XM_005260808.4; XM_017028021.1; XM_017028022.1; XM_017028023.1 CD39ectonucleoside NM_001098175.1; NM_001164178.1; triphosphateNM_001164179.1; NM_001164181.1; diphosphohydrolase 1 NM_001164182.1;NM_001164183.1; NM_001312654.1; NM_001320916.1; NM_001776.5;XM_011540370.2; XM_011540371.2; XM_011540372.2; XM_011540373.2;XM_011540374.2; XM_011540376.2; XM_011540377.2; XM_017016958.1;XM_017016959.1; XM_017016960.1; XM_017016961.1; XM_017016962.1;XM_017016963.1; XM_017016964.1 CD73 5′-nucleotidase ecto NM_001204813.1;NM_002526.3 MIF macrophage migration NM_002415.1 inhibitory factor(glycosylation-inhibiting factor) VEGF vascular endothelial growthNM_001025366.2; NM_001025367.2; factor A NM_001025368.2; NM_001025369.2;NM_001025370.2; NM_001033756.2; NM_001171622.1; NM_001171623.1;NM_001171624.1; NM_001171625.1; NM_001171626.1; NM_001171627.1;NM_001171628.1; NM_001171629.1; NM_001171630.1; NM_001204384.1;NM_001204385.1; NM_001287044.1; NM_001317010.1; NM_003376.5; PIGFphosphatidylinositol glycan NM_002643.3; NM_173074.2; XM_005264369.2;anchor biosynthesis class F XM_011532908.2 CXCR4 C-X-C motif chemokineNM_001008540.2; NM_001348056.1; receptor 4 NM_001348059.1;NM_001348060.1; NM_003467.2 CCR2 C-C motif chemokine NM_001123041.2;NM_001123396.1; receptor 2 XM_011534069.1 PLK1 polo like kinase 1NM_005030.5 MTDH metadherin NM_178812.3; XM_005251099.3; XM_011517367.2;XM_011517368.2; XM_011517369.2; XM_011517370.2; XM_017013966.1;XM_017013967.1; XM_017013968.1

Such embodiments are not limited to a particular manner of assessing thedelivery profile of the siRNA in vitro and/or in vivo. In someembodiments, labelling the siRNA molecules with an imaging agent (e.g.,fluorescent dye FITC, RITC, Cy™ dyes, Dylight® dyes, Alexa Fluor® dyes,or lanthanide probes) or a radiotracer permits visualization of thebiodistribution of siRNA molecules at the organ level and also theintracellular delivery profile. In some embodiments, RT-PCR and westernblot are used to analyze the target protein at the mRNA level andprotein level, respectively.

In certain embodiments, the present invention provides methods forinhibiting a target gene in a cell comprising introducing into the cell(associated with an therapeutic construct) an siRNA capable ofinhibiting the target gene by RNA interference, wherein the siRNAcomprises two RNA strands that are complementary to each other, whereinthe siRNA is loaded onto a therapeutic construct. In some embodiments,the siRNA is modified with cholesterol at the 3′ sense strand. In someembodiments, the cell is within a human being or an animal subject(e.g., horses, dogs, cats, or other domestic, farm, or other animalswith cancer).

MicroRNAs (miRNAs) or miRNA mimics are short, non-coding RNAs that cantarget and substantially silence protein coding genes through 3′-UTRelements. Important roles for miRNAs in numerous biological processeshave been established, but comprehensive analyses of miRNA function incomplex diseases are lacking. miRNAs are initially transcribed asprimary miRNAs (pri-miRNAs) that are then cleaved by the nuclear RNAsesDrosha and Pasha to yield precursor-miRNAs (pre-miRNAs). Theseprecursors are further processed by the cytoplasmic RNAse III dicer toform short double stranded miR-miR* duplexes, one strand of which (miR)is then integrated into the RNA Induced Silencing Complex (RISC) thatincludes the enzymes dicer and Argonaute (Ago). The mature miRNAs(˜17-24 nt) direct RISC to specific target sites located within the3′UTR of target genes. Once bound to target sites, miRNAs repressestranslation through mRNA decay, translational inhibition and/orsequestration into processing bodies (P-bodies) (Eulalio et al., Cell,132:9-14, 2008; Behm-Ansmant et al., Cold Spring Harb. Symp. Quant.Biol., 71:523-530, 2006; Chu and Rana, Plos. Biology., 4:e210, 2006).Recent estimates find that over 60% of protein coding genes carry 3′-UTRmiRNA target sites (Friedman et al., Genome Res., 19:92-105, 2009). Inthis regard, miRNAs act as key regulators of processes as diverse asearly development (Reinhart et al., Nature, 403:901-906, 2000), cellproliferation and cell death (Brennecke et al., Cell, 113(1):25-36,2003), apoptosis and fat metabolism (Xu et al., Curr. Biol.,13(9):790-795, 2003), and cell differentiation (Chen et al., Mol.Microbiol., 53843-856, 2004; Dostie et al., RNA-A Publication of the RNASociety, 9:180-186, 2003). In addition, studies of miRNA expression inchronic lymphocytic leukemia (Calin et al., Proc. Natl. Acad. Sci. USA,105:5166-5171, 2008), colonic adenocarcinoma (Michael et al., Mol.Cancer Res., 1:882-891, 2003), Burkitt's lymphoma (Metzler et al., GenesChromosomes Cancer, 39:167-169, 2004), cardiac disease (Zhao et al.,Cell, 129:303-317, 2007) and viral infection (Pfeffer et al., Science,304:734-736, 2004) suggest vital links between miRNA and numerousdiseases.

miRNAs thus far observed have been approximately 21-22 nucleotides inlength and they arise from longer precursors, which are transcribed fromnon-protein-encoding genes. Reviewed in Carrington and Ambros (Science,301(5631):336-338, 2003). The precursors form structures that fold backon each other in self-complementary regions; they are then processed bythe nuclease Dicer in animals (or DCL1 in plants). miRNA moleculesinterrupt translation through precise or imprecise base-pairing withtheir targets. In some embodiments, a miRNA may be used as a componentof a provided therapeutic construct, therapeutically, or administered toa subject, such as a human patient, to treat a disease such as, e.g.,cancer; alternately, in some embodiments, a nucleic acid that iscomplementary to the miRNA may be therapeutically administered to asubject in vivo or used in vitro to generate the desired therapeuticmiRNA (e.g., miRNA-142-3p, miRNA-142-3p, miRNA-124, or miRNA-138). Inthis way, the complementary nucleic acid may be used as a template togenerate the desired therapeutic miRNA (e.g., miRNA-142-3p,miRNA-142-3p, miRNA-124, or miRNA-138).

Additional Anti-Cancer Agent(s).

The phrase “anti-cancer agent” is used in accordance with its plainordinary meaning and refers to a composition (e.g. compound, drug,antagonist, inhibitor, modulator) having antineoplastic properties orthe ability to inhibit the growth or proliferation of cells. In someembodiments, an anti-cancer agent is a chemotherapeutic agent. In someembodiments, an anti-cancer agent is a targeted therapeutic agent. Insome embodiments, an anti-cancer agent is an immune checkpointinhibitor. In some embodiments, an anti-cancer agent is an agentidentified herein having utility in methods of treating cancer. In someembodiments, an anti-cancer agent is an agent approved by the FDA orsimilar regulatory agency of a country other than the USA, for treatingcancer.

Examples of anti-cancer agents include, but are not limited to, MEK(e.g., MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g., XL518, CI-1040,PD035901, selumetinib/AZD6244, GSK1120212/trametinib, GDC-0973,ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733,PD318088, AS703026, BAY 869766, PD184352, SB239063, BAY 43-9006);alkylating agents such as nitrogen mustards (e.g., mechloroethamine,cyclophosphamide, uramustine, chlorambucil, melphalan, ifosfamide),ethylenimine and methylmelamines (e.g., hexamethlymelamine andthiotepa), alkyl sulfonates (e.g., busulfan and hepsulfam), nitrosoureas(e.g., carmustine, lomusitne, semustine, and streptozocin), andtriazenes (e.g., decarbazine); anti-metabolites such as folic acidanalogs (e.g., methotrexate, leucovorin, raltitrexed, and pemetrexed),pyrimidine analogs (e.g., fluorouracil, floxouridine, cytarabine,capecitabine, and gemcitabine), and purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin, fludarabine, and5-azathioprine); plant alkaloids (e.g., vincristine, vinblastine,vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, andhomoharringtonine); topoisomerase inhibitors such as camptothecinderivatives (e.g., irinotecan and topotecan), amsacrine, andepipodophyllotoxins (e.g., etoposide (VP16), etoposide phosphate, andteniposide); antibiotics such as anthracenediones (e.g., mitoxantrone),anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, andfluorodaunorunicin hydrochloride), streptomyces-derived antibiotics orderivatives thereof (e.g., dactinomycin, bleomycin, mitomycin,geldanamycin, plicamycin, and 17-N-allylamino-17-demethoxygeldanamycin(17-AAG; tanespimycin), clofazimine, and beta lactam derivatives;platinum-based compounds (e.g., cisplatin, oxaliplatin, carboplatin);substituted urea (e.g., hydroxyurea); methyl hydrazine derivative (e.g.,procarbazine), adrenocortical suppressant (e.g., mitotane andaminoglutethimide); angiogenesis-inhibiting enzymes (e.g.,L-asparaginase and arginine deiminase); Pl3K inhibitors (e.g.,wortmannin and LY294002); mTOR inhibitors (e.g., sertraline); DNAmethyltransferase inhibitors (e.g., 5-aza-2′-deoxycytidine); antisenseoligonucleotides; apoptosis gene modulators; apoptosis regulators (e.g.,deoxyadenosine and triptolide); BCR/ABL antagonists; bFGF inhibitor;casein kinase inhibitors (ICOS); gallium nitrate; gelatinase inhibitors;glutathione inhibitors (e.g., etanidazole); immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; leukemia inhibitingfactor; matrilysin inhibitors; matrix metalloproteinase inhibitors; MIFinhibitor; mismatched double stranded RNA; mycobacterial cell wallextract; nitric oxide modulators; phosphatase inhibitors; plasminogenactivator inhibitor; proteasome inhibitors (e.g., bortezomib); proteinA-based immune modulator; protein kinase C inhibitors; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors; rasfarnesyl protein transferase inhibitors; ras inhibitors; ras-GAPinhibitor; ribozymes; signal transduction inhibitors/modulators (e.g.,itraconazole); single chain antigen-binding protein; stem cellinhibitor; stem-cell division inhibitors; stromelysin inhibitors;synthetic glycosaminoglycans; telomerase inhibitors; thyroid stimulatinghormones; translation inhibitors; urokinase receptor antagonists;gonadotropin-releasing hormone agonists (GnRH) such as goserelin andleuprolide (leuprorelin); steroids such as adrenocorticosteroids (e.g.,prednisone and dexamethasone); progestins (e.g., hydroxyprogesteronecaproate, megestrol acetate, medroxyprogesterone acetate);antiprogestrogens (e.g., mifepristone); estrogens (e.g.,di-ethlystilbestrol and ethinyl estradiol); antiestrogens such asaromatase inhibitors (e.g., exemestane, fadrozole, letrozole,pentrozole, and anastrozole), selective estrogen receptor modulators(e.g., tamoxifen, tamoxifen methiodide, panomifene, and clomifeneanalogues); androgens (e.g., testosterone propionate andfluoxymesterone); antiandrogen (e.g., flutamide, finasteride, andbicalutamide); immunostimulants such as levamisole, interleukins (e.g.,interleukin-2) and interferons/interferon agonists (e.g.,alpha-interferon); monoclonal antibodies such as anti-CD20 (e.g.,rituximab), anti-HER2 (e.g., trastuzumab), anti-CD52, anti-CD25 (e.g.,daclizumab), anti-HLA-DR, and anti-VEGF monoclonal antibodies);immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicinconjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate,etc.); radio immunotherapeutic agents (e.g., anti-CD20 monoclonalantibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I, etc.); statins (e.g.,cerivastatin and pitavastatin); 5-T1_(B) receptor agonists (e.g.,5-nonyloxytryptamine); BRAF kinase inhibitors (e.g., vemurafenib anddabrafenib); tyrosine kinase inhibitors such as inhibitors of one ormore of EGFR, HER2, KDR, FLT4, EphB4, and Src (e.g., gefitinib(Iressa™), erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib(Tykerb™), panitumumab (Vectibix™) vandetanib (Caprelsa™),afatinib/BIBW2992, CI-1033/canertinib, neratinib/HKI-272, CP-724714,TAK-285, AST-1306, ARRY334543, AG-1478, dacomitinib/PF299804,OSI-420/desmethyl erlotinib, AZD8931, ARRY-380, AEE788,pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647,PD153035, BMS-599626, sorafenib, imatinib (Gleevec®), sunitinib, anddasatinib; immune-checkpoint inhibitors (e.g., anti-CTLA-4, anti-PD1/L1antibodies); PLK1 inhibitors (GSK461364, BI2536, Tak960, NMS-P937,B16727), mitotic kinase inhibitors, or the like, or mixtures thereof(e.g., leuprolide+estrogen+progesterone).

Additionally, the therapeutic constructs described herein can beco-administered with conventional immunotherapeutic agents including,but not limited to, immunostimulants (e.g., Bacille Calmette-Guerin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), therapeuticmonoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52,anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g.,anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22monoclonal antibody-pseudomonas exotoxin conjugate, etc.),immune-checkpoint inhibitors (e.g., anti-CTLA4, anti-PD-1, anti-PD-L1antibodies), and radioimmunotherapy (e.g., anti-CD20 monoclonal antibodyconjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I, etc.). These immunotherapeutic agentscan also be loaded directly onto the therapeutic constructs to enhancetheir therapeutic effect, reduce toxicity, and reduce administrationtime.

In a further embodiment, the therapeutic constructs described herein canbe co-administered with conventional radiotherapeutic agents including,but not limited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y,⁸⁷Y, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At and ²¹²Bi. These radiotherapeutic agents can also beloaded directly onto the therapeutic constructs to enhance thetherapeutic effect, reduce toxicity, and reduce administration time.

Targeting Moieties

One or more targeting moieties (a.k.a., targeting molecules) can beloaded into, attached to the surface of, and/or enclosed within thedelivery vehicle. In embodiments, the targeting moiety is displayed onthe exterior surface of the delivery vehicle. Such targeting moietiesmay be particularly beneficial for systemic delivery.

Exemplary target molecules include proteins, peptides, ligands, nucleicacids, lipids, saccharides, or polysaccharides that bind to one or moretargets associated with an organ, tissue, cell, or extracellular matrix,or specific type of tumor or infected cell. The degree of specificitywith which the delivery vehicles are targeted can be modulated throughthe selection of a targeting molecule with the appropriate affinity andspecificity. For example, antibodies are very specific. These can bepolyclonal, monoclonal, fragments, recombinant, or single chain, many ofwhich are commercially available or readily obtained using standardtechniques. T-cell specific molecules, antigens, and tumor targetingmolecules can be bound to the surface of the therapeutic constructs. Thetargeting molecules may be conjugated to the terminus of one or more PEGchains present on the surface of the particle.

In some embodiments, the targeting moiety is an antibody or antigenbinding fragment (e.g., single chain variable fragments) thereof thatspecifically recognizes a cell or tumor marker that is presentexclusively or in elevated amounts on a target cell, such as a malignantcell (e.g., a tumor antigen). Suitable targeting molecules that can beused to direct therapeutic constructs to cells and tissues of interest,as well as methods of conjugating target molecules to nanoparticles, areknown in the art.

See, for example, Ruoslahti et al. (Nat. Rev. Cancer, 2:83-90, 2002). Incertain cases, therapeutic agents can be toxic to both cancer and immunecells, resulting in suboptimal efficacy. Thus, in certain embodiments,therapeutic constructs can be conjugated with a targeting moiety toenrich the delivery of at least one mitotic kinase inhibitor and atleast one immune checkpoint inhibitor to only cancer cells. Examplesnonexclusively include antibodies against HER2, EGFR, PD-L1, etc. thatare overexpressed on cancer cells. In some embodiments, therapeuticconstructs can be conjugated with a targeting moiety to enrich thedelivery of at least one mitotic kinase inhibitor and at least oneimmune checkpoint inhibitor to only immune cells.

Targeting molecules can also include neuropilins and endothelialtargeting molecules, integrins, selectins, adhesion molecules, bonetargeting molecules such as zoledronic acid and alendronic acid (e.g.,to target cancer metastasized to bone), stroma, and fibroblast targetingmolecules.

In some embodiments, the targeting moiety targets the therapeuticconstruct to antigen-presenting cells (APCs), and particularly to asubclass of APCs known as dendritic cells. Dendritic cells express anumber of cell surface receptors that can mediate endocytosis. In someembodiments, therapeutic construct enhances the activity of DC toprocess tumor antigen. Targeted delivery to DC may be performed.Targeting exogenous antigens to internalizing surface molecules onsystemically-distributed antigen presenting cells facilitates uptake ofthe particle and can overcomes a major rate-limiting step in thetherapy.

Dendritic cell targeting molecules include monoclonal or polyclonalantibodies or fragments thereof that recognize and bind to epitopesdisplayed on the surface of dendritic cells. Dendritic cell targetingmolecules also include ligands which bind to a cell surface receptor ondendritic cells. One such receptor, the lectin DEC-205, has been used invitro and in mice to boost both humoral (antibody-based) and cellular(CD8 T cell) responses by 2-4 orders of magnitude (Hawiger et al., J.Exp. Med., 194(6):769-79, 2001; Bonifaz et al., J. Exp. Med.,196(12):1627-38 2002; Bonifaz et al., J. Exp. Med., 199(6):815-24,2004). In these reports, antigens were fused to an anti-DEC205 heavychain and a recombinant antibody molecule was used for immunization.

A variety of other endocytic receptors, including a mannose-specificlectin (mannose receptor) and IgG Fc receptors, have also been targetedin this way with similar enhancement of antigen presentation efficiency.Other suitable receptors which may be targeted include, but are notlimited to, DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, heat shock proteinreceptors and scavenger receptors. Targeting moieties for thesereceptors can be attached to the therapeutic constructs for theirpreferential uptake into immune cells that express these receptors.Example is mannose attached on the therapeutic constructs for targeteddelivery to macrophages and DCs that have high levels of mannosereceptors.

Other receptors which may be targeted include the toll-like receptors(TLRs). TLRs recognize and bind to pathogen-associated molecularpatterns (PAMPs). PAMPs target the TLR on the surface of the dendriticcell and signals internally, thereby potentially increasing DC antigenuptake, maturation and T-cell stimulatory capacity. PAMPs conjugated tothe particle surface or co-encapsulated include unmethylated CpG DNA(bacterial), double-stranded RNA (viral), lipopolysaccharide(bacterial), peptidoglycan (bacterial), lipoarabinomannin (bacterial),zymosan (yeast), mycoplasmal lipoproteins such as MALP-2 (bacterial),flagellin (bacterial) poly(inosinic-cytidylic) acid (bacterial),lipoteichoic acid (bacterial) or imidazoquinolines (synthetic).

Targeting molecules can be covalently bound to delivery vehicles using avariety of methods known in the art. In preferred embodiments thetargeting moiety is attached to the delivery vehicle by PEGylation or abiotin-avidin bridge.

CD40 Agonist. In a particular embodiment, the targeting moiety targetsCD40. The moiety can be a CD40 agonist. The cell surface molecule CD40is a member of the tumor necrosis factor receptor superfamily and isbroadly expressed by immune, hematopoietic, vascular, epithelial, andother cells, including a wide range of tumor cells. As a potentialtarget for cancer therapy, CD40 may mediate tumor regression throughboth an indirect effect of immune activation and a direct cytotoxiceffect on the tumor, resulting in a “two-for-one” mechanism of action ofCD40 agonists. CD40 agonists are known in the art and reviewed inVonderheide (Clin Cancer Res, 13(4):1083-1088, 2007). Exemplary agonistsinclude recombinant CD40L (recombinant human trimer), CD-870, 893 (fullyhuman IgG2 mAb), SGN-40 (humanized IgG1), and HCD 122 (fully human IgG1mAb). Soluble agonistic CD40 antibodies have been shown to substitutefor T-cell help provided by CD4+lymphocytes in murine models of Tcell-mediated immunity (Khalil et al., Update Cancer Ther., 2:61-65,2007).

Integrin Ligand. In another embodiment, the targeting moiety is a ligandfor an integrin. Studies show that integrins are overexpressed on thesurface of tumor cells and can thus serve as a marker that distinguishesbetween tumor cells and normal cells. Certain integrins also activateTGF-β through an extracellular pathway. After latent TGF-β is releasedfrom a tumor cell, it binds with integrin on the surface of the tumorcell, leading to the activation of the latent TGF-β. IncreasedTGF-βconcentrations in the tumor microenvironment support immunesuppression and recruit regulatory T cells to the tumor environment.

RGD peptide can serve a dual function: it is not only a typicalintegrin-targeting ligand (Ruoslahti et al., Annu. Rev. Cell Dev. Biol.,12:697-715, 1996) but also serves as an immune danger signal, activatingAPCs (Altincicek et al., Biol Chem., 390, 1303-11, 2009). Therefore, ina preferred embodiment, RGD peptide is loaded into, attached to thesurface of, and/or enclosed within the delivery vehicle.

T Cell Receptor that Recognizes the p53 Antigen. In a particularembodiment, the targeting moiety is a T cell receptor (TCR) thatrecognizes the p53 antigen within the context of human MHC. T cellreceptor recombinant proteins derived from bacterial, eukaryotic oryeast cells include T cell receptors composed of the alpha, beta chainsor gamma/delta chains (α/β TCR or γ/Δ TCRs).

IL-15/IL-15Rα. In another embodiment, the targeting moiety is anIL-15/IL-15Rα complex. Interleukin-15 (IL-15) is a cytokine that sharescertain receptor subunits with IL-2 and thus has some overlappingmechanisms of action. IL-15 is expressed by dendritic cells and providesa critical signal for the proliferation and priming of natural killer(NK) cells. Accordingly, IL-15/IL-15Rα complex can be used to targetnanoparticulate compositions to, for example, natural killer (NK) cells.

(V) DELIVERY SYSTEMS

Embodiments of the herein-provided therapeutic constructs are agnosticas to the delivery system employed for co-delivery of at least onemitotic kinase inhibitor and at least one immune checkpoint inhibitor.Thus, in various embodiments, the delivery system can use or be based onany type of known or to-be-developed particulate delivery vehicle. Theseinclude nanoparticles, fullerenes, endohedral metallofullerenes,trimetallic nitride templated endohedral metallofullerenes,single-walled and multi-walled carbon nanotubes, branched and dendriticcarbon nanotubes, gold nanorods, silver nanorods, single-walled andmulti-walled boron/nitrate nanotubes, calcium phosphate particles,aluminum salt particles, carbon nanotube peapods, carbon nanohorns,carbon nanohorn peapods, liposomes, lipid-based nanoparticles, lipoplex,polymeric nanoparticles, polyplex, nanoshells, dendrimers,microparticles, quantum dots, superparamagnetic nanoparticles, nanorods,cellulose nanoparticles, glass and polymer micro- and nano-spheres,biodegradable PLGA micro- and nano-spheres, gold nanoparticles, silvernanoparticles, carbon nanoparticles, iron nanoparticles, porous andnon-porous silica nanoparticles, and modified micelles. Hybrid particlesthat consist of several classes of materials can also be used. Particlesin nanometer and micron sizes can be used. Therapeutic agents,adjuvants, and any additional compounds can be included with thedelivery agent by any suitable means, e.g., loaded into, attached to thesurface of, coupled to, enclosed within, or contained within thedelivery system. Such agents may be encapsulated, covalently bound, ornon-covalently bound (e.g., by electrostatic, hydrophobic, van derWaals, or compound-specific interaction (such as nucleic acid basepairing, ligand-receptor, antibody-antigen, biotin-avidin, etc.))

In some embodiments, the delivery system includes a mesoporous silicananoparticle (MSNP), such as those described in U.S. Patent PublicationNo. US2017/0172923 and No. 2017/0173169, the MSNPs of which are herebyincorporated by reference.

In some embodiments, the mean particle size of the mesoporousnanoparticle (or a different nanoparticle) is about 5 nm to about 200nm, about 5 nm to about 90 nm, about 5 nm to about 20 nm, about 30 nm toabout 100 nm, about 30 nm to about 80 nm, about 30 nm to about 60 nm,about 40 nm to about 80 nm, about 70 nm to about 90 nm, or about 5 nm,about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm.

In some embodiments, the mesoporous silica nanoparticle is coated withcationic polymers or other compounds. The cationic polymer may bind tothe surface of the nanoparticle using any appropriate means. In someembodiments, the cationic polymer binds to the nanoparticle viaelectrostatic interaction. The cationic polymer may be any polymer witha positive charge, such as, but not limited to, PEI, polyamidoamine,poly(allylamine), poly(diallyldimethylammonium chloride), chitosan,poly(N-isopropyl acrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid), poly(L-lysine), diethylaminoethyl-dextran,poly-(N-ethyl-vinylpyridinium bromide), poly(dimethylamino)ethylmethacrylate), or poly(ethyleneglycol)-co-poly(trimethylaminoethylmethacrylate chloride). Othercationic polymers will be apparent to those of skill in the art, and maybe found, for example, in Polymer Handbook, 4th Edition, Edited by:Brandrup, E. H. Immergut, and E. A. Grukle; John Wiley & Sons, 2003).

The cationic polymers may be linear or branched. In some embodiments,the cationic polymers may range in size from about 500 Da to 25 kDa andmay be branched or linear. For example, branched PEI with an averagesize of 1.8 kDa to 10 kDa may be loaded onto the nanoparticle core. Theratio of cationic polymer to nanoparticle may be varied depending on thedesired result. The cationic polymer may be present at 1 to 50 wt. % ofthe nanoconstruct, e.g., 5 to 40 wt. %, 10 to 30 wt. %, 20 to 30 wt. %,5 to 15 wt. %, 5 to 20 wt. %, 5 to 25 wt. %, 5 to 30 wt. %, 10 to 20 wt.%, 10 to 25 wt. %, or 25 to 40 wt. %, e.g., about 5, 10, 15, 20, 25, 30,or 35 wt. %. In some embodiments, the cationic polymer is present at 10to 20 wt. %.

In some embodiments, the cationic polymer is crosslinked, e.g., with acleavable disulfide bond, pre- or post-coating on the nanoparticle. Insome embodiments, the attached cationic polymer is crosslinked afterbinding to the nanoparticles, e.g., MSNP, using, for example, DSP(dithiobis[succinimidyl propionate]), DTSSP(3,3′-dithiobis(sulfosuccinimidyl propionate), and DTBP (dimethyl3,3′-dithiobispropionimidate). The crosslinking may occur in the absenceor presence of free cationic polymer in solution. In other embodiments,the cationic polymer is not crosslinked.

A stabilizer may be conjugated to the MSNP (or a different nanoparticle)and/or the cationic polymer, e.g., by any appropriate means. In someembodiments, a stabilizer is conjugated to an amine or other reactivegroup of a cross-linked cationic polymer coated on the nanoparticle(e.g., a MSNP). Exemplary stabilizers include, but are not limited to,PEG, dextran, polysialic acid, hyaluronic acid, polyvinyl pyrrolidone,polyvinyl alcohol, and polyacrylamide, or a combination thereof.

A stabilizer may have multiple chemically reactive groups, e.g., forattachment to the nanoparticle, cationic polymer, and/or othercomponent. For example, a reactive stabilizer, e.g., a PEG derivative,may have two electrophilic moieties, such asmaleimide-PEG-N-hydroxysuccinimidyl ester (Mal-PEG-NHS), which containsboth a Michael acceptor and an activated ester. The stabilizer, e.g.,PEG, used in conjunction with the compositions and methods of theinvention generally has a molecular weight ranging between 500 Da-40kDa, e.g., 2-10 kDa. The stabilizer may be present at 1 to 50 wt. % ofthe nanoconstruct, e.g., 5 to 30 wt. %, 10 to 20 wt. %, 10 to 25 wt. %,5 to 15 wt. %, 5 to 20 wt. %, 5 to 25 wt. %, or 1 to 10 wt. %, e.g.,about 5, 10, 15, 20, 25, 35, 40 or 45 wt. %.

“Mean particle size” as used herein, generally refers to the statisticalmean particle size (diameter) of the particles in a population ofparticles. The diameter of an essentially spherical particle may referto the physical or hydrodynamic diameter. The diameter of anon-spherical particle may refer preferentially to the hydrodynamicdiameter. As used herein, the diameter of a non-spherical particle mayrefer to the largest linear distance between two points on the surfaceof the particle. Mean hydrodynamic particle size can be measured usingmethods known in the art, such as dynamic light scattering.

“Monodisperse” and “homogeneous size distribution”, are usedinterchangeably herein and describe a population of nanoparticles ormicroparticles where all of the particles are the same or nearly thesame size. As used herein, a monodisperse distribution refers toparticle distributions in which 90% of the distribution lies within 15%of the median particle size, more preferably within 10% of the medianparticle size, most preferably within 5% of the median particle size.

“Nanoparticle”, as used herein, generally refers to a particle having adiameter from about 5 nm up to, but not including, about 1 micron,preferably from 20 nm to about 1 micron. The particles can have anyshape. Nanoparticles having a spherical shape are generally referred toas “nanospheres”. The present invention is not limited to specific typesor kinds of nanoparticles for complexing with at least one mitotickinase inhibitor and at least one immune checkpoint inhibitor configuredfor treating or preventing cancer and related hyperproliferativedisorders.

Examples of nanoparticles include fullerenes (a.k.a. C₆₀, C₇₀, C₇₆, C₈₀,C₈₄), endohedral metallofullerenes (EMI's), which contain additionalatoms, ions, or clusters inside their fullerene cage), trimetallicnitride templated endohedral metallofullerenes (TNT EMEs, high-symmetryfour-atom molecular cluster endohedrals, which are formed in atrimetallic nitride template within the carbon cage), single-walled andmulti-walled carbon nanotubes, branched and dendritic carbon nanotubes,gold nanorods, silver nanorods, single-walled and multi-walledboron/nitrate nanotubes, carbon nanotube peapods (nanotubes withinternal metallo-fullerenes and/or other internal chemical structures),carbon nanohorns, carbon nanohorn peapods, lipid particles liposomes,lipoplex, polymeric nanoparticles, polyplex, nanoshells, dendrimers,quantum dots, superparamagnetic nanoparticles, nanorods, and cellulosenanoparticles. Other exemplary nanoparticles include glass and polymermicro- and nano-spheres, biodegradable PLGA micro- and nano-spheres,gold, silver, platinum, carbon, and iron nanoparticles.

In some embodiments, the nanoparticle is a modified micelle. In theseembodiments, the modified micelle comprises polyol polymers modified tocontain a hydrophobic polymer block. The term “hydrophobic polymerblock” as used in the present disclosure indicates a segment of thepolymer that on its own would be hydrophobic. The term “micelle” as usedherein refers to an aggregate of molecules dispersed in a liquid. Atypical micelle in aqueous solution forms an aggregate with thehydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic single tail regions in the micelle center.In some embodiments the head region may be, for example, a surfaceregion of the polyol polymer while the tail region may be, for example,the hydrophobic polymer block region of the polyol polymer.

The invention further encompasses use of particles on the micrometerscale in addition to the nanometer scale. Where microparticles are used,it is preferred that they are relatively small, on the order of 1-50micrometers. For ease of discussion, the use herein of “nanoparticles”encompasses true nanoparticles (sizes of from about 1 nm to about 1000nm), microparticles (e.g., from about 1 micrometer to about 50micrometers), or both.

Examples of nanoparticles include, by way of example and withoutlimitation, paramagnetic nanoparticles, superparamagnetic nanoparticles,metal nanoparticles, fullerene-like materials, inorganic nanotubes,dendrimers, dendrimers with covalently attached metal chelates,nanofibers, nanohorns, nano-onions, nanorods, nanoropes, and quantumdots. In some embodiments, a nanoparticle is a metal nanoparticle (forexample, a nanoparticle of gold, palladium, platinum, silver, copper,nickel, cobalt, iridium, or an alloy of two or more thereof).Nanoparticles can include a core or a core and a shell, as in core-shellnanoparticles. Hybrid particles that consist of several classes ofmaterials can also be used.

Therapeutic construct-containing compositions including at least onemitotic kinase inhibitor and at least one immune checkpoint inhibitoreach loaded into, attached to the surface of, and/or enclosed within adelivery vehicle, are disclosed. The nanoparticulate compositions offera number of advantages over delivering the active agent or agents to thetarget cells in solution. For example, the nanoparticulate compositionspresent a localized concentration of the one or more active agents on orin a nanoparticle leading to increased avidity when the nanoparticleencounters the target cells. The nanoparticulate compositions can alsoserve as a depot of active agent with tunable release kinetics that canextend over several days to prolong effective systemic half-life andefficacy of the agent or agents.

Typically, two or more active agents (including at least one mitotickinase inhibitor and at least one immune checkpoint inhibitor) areloaded into, attached to the surface of, and/or enclosed within adelivery vehicle. The relative concentrations of each of the two or moreactive agents and their location on or within the delivery vehicle canbe manipulated during manufacture of the compositions to adapt apreferred dosage and presentation that will be received by the targetcell. Loading of two or more active agents into or onto the samedelivery vehicle allows the two or more active agents to be presented tothe target cell or same tumor microenvironment simultaneously or in anotherwise predetermined order to the target cell.

The delivery vehicles can be, for example, nanolipogels, polymericparticles, silica particles, liposomes, or multilamellar vesicles. Inthe certain embodiments, the particulate delivery vehicles are nanoscalecompositions, for example, 10 nm up to, but not including, about 1micron. However, it will be appreciated that in some embodiments, andfor some uses, the particles can be smaller, or larger (e.g.,microparticles, etc.). Although example therapeutic constructs disclosedherein may be referred to nanoparticulate compositions, it will beappreciated that in some embodiments and for some uses the particulatecompositions can be somewhat larger than nanoparticles. For example,particulate compositions can also be between about 1 micron to about1000 microns. Such compositions can be referred to as microparticulatecompositions.

In embodiments for treating cancer it is desirable that the particle beof a size suitable to access the tumor microenvironment. In particularembodiments, the particle is of a size suitable to access the tumormicroenvironment and/or the tumor cells by enhanced permeability andretention (EPR) effect. EPR refers to the property by which certainsizes of molecules (e.g., the particulate compositions discussed herein)tend to accumulate in tumor tissue much more than they do in normaltissues. Therefore, in compositions for treatment of cancer, thedelivery vehicle is preferably in the range of about 25 nm to about 500nm inclusive, more preferably in the range of about 30 nm to about 300nm inclusive.

Nanolipogels. Nanolipogels are core-shell nano-particulates that combinethe advantages of both liposomes and polymer-based particles forsustained delivery of active agents. In some of these embodiments andapplications nanolipogels can exhibit, increased loading efficiency,increased sustained release, and improved therapeutic efficacy forcombinations of macromolecules and molecules compared to conventionalnanoparticle compositions.

Typically, the outer shell of the nanolipogel protects cargo and,provides biocompatibility as well as a surface for functionalizationwith targeting molecule(s). The outer shell encapsulates components sothey are not exposed until desired, for example, in response toenvironmental conditions or stimuli, creating monodisperse, reproducibleparticle populations, and mediating internalization into desired celltypes. The inner core, which can be a dendrimer or other polymer, hasseparate and additive functionalities to the outer shell. For example,the inner shell allows for secondary deposition of drug, vaccine, orimaging agent; increases loading of components with differentphysiochemical properties into the particle; allows for tunable releaseof contents from particles; increases cytosolic availability of DNA/RNA,drug, and/or protein by disrupting endosomes, all leading to enhanceddrug effects, antigen presentation, and transfection/silencing.

Nanolipogels have a polymer matrix core containing one or more hostmolecules. The polymeric matrix is preferably a hydrogel, such as acrosslinked block copolymer containing one or more poly(alkylene oxide)segments, such as polyethylene glycol, and one or more aliphaticpolyester segments, such as polylactic acid. One or more cargo moleculesis dispersed within or covalently bound to the polymeric matrix. Thehydrogel core is surrounded by a liposomal shell.

Nanolipogels can be constructed to incorporate a variety of activeagents that can subsequently be released in a controlled fashion. Activeagents can be dispersed within the hydrogel matrix, dispersed within theliposomal shell, covalently attached to the liposomal shell, andcombinations thereof. Active agents can be selectively incorporated ateach of these locales within the nanolipogel. Furthermore, the releaserate of active agents from each of these locales can be independentlytuned. Because each of these locales possesses distinct properties,including size and hydrophobicity/hydrophilicity, the chemical entitiesindependently incorporated at each of these locales can differdramatically with respect to size and composition. For example,nanolipogels can be loaded with one or more compounds dispersed withinthe polymeric matrix as well as at least one mitotic kinase inhibitorand at least one immune checkpoint inhibitor. Nanolipogels can be loadedprovide simultaneous sustained release of agents that differ widely inchemical composition and molecular weight.

Nanolipogels are typically spherical in shape, with average particlesizes ranging between about 50 nm and about 1000 nm, more preferablybetween about 75 nm and about 300 nm, most preferably between about 90nm and about 200 nm. In certain embodiments, the nanolipogels possess anaverage particle size between about 100 nm and about 140 nm. Particlesmay be non-spherical.

Depending upon the nature of the lipids present in the liposomal shellof the nanolipogels, nanolipogels having a positive, negative, or nearneutral surface charge may be prepared. In certain embodiments, thenanolipogels possess a near neutral surface charge. In certainembodiments, the nanolipogels possess a ζ-potential of between about 10mV and about −10 mV, more preferably between about 5 mV and about −5 mV,more preferably between about 3 mV and about −3 mV, most preferablybetween about 2 mV and about −2 mV.

Hydrophobic active agents, such as proteins, may be covalently connectedto the surface of the nanolipogel, whereas hydrophilic active agents maybe covalently connected to the surface of the nanolipogel or dispersedwithin the liposomal shell. In certain embodiments, the liposomal shellincludes one or more PEGylated lipids. In these cases, one or moreactive agents may be conjugated to the terminus of one or more PEGchains present on the surface of the liposomal shell.

In another embodiment, the lipid is modified to include an avidinmoiety, enabling a biotinylated targeting moiety, detectable label, orother active agent to be coupled thereto, if so desired.

In particular embodiments, one or more active agents are covalentlyconnected to the surface of the nanolipogel via a linking group that iscleaved in response to an external chemical or physical stimulus, suchas a change in ambient pH, so as to trigger release of the active agentat a desired physiological locale.

Core. The nanolipogel core is formed from a polymeric matrix. The matrixcan include one or more host molecules as discussed in more detailbelow. The nanolipogel core may further include one or more activeagents. The active agents may be complexed to a host molecule, dispersedwith polymeric matrix, or combinations thereof.

The polymeric matrix of the nanolipogels may be formed from one or morepolymers or copolymers. By varying the composition and morphology of thepolymeric matrix, one can achieve a variety of controlled releasecharacteristics, permitting the delivery of moderate constant doses ofone or more active agents over prolonged periods of time.

The polymeric matrix may be formed from non-biodegradable orbiodegradable polymers; however, preferably, the polymeric matrix isbiodegradable. The polymeric matrix can be selected to degrade over atime period ranging from one day to one year, more preferably from sevendays to 26 weeks, more preferably from seven days to 20 weeks, mostpreferably from seven days to 16 weeks. Biodegradable cross-linkers maybe used to increase molecular weight of polymers, which are clearablefrom the body as small fragments after degradation of the cross-linkers.

In general, synthetic polymers are preferred, although natural polymersmay be used. Representative polymers include poly(hydroxy acids) such aspoly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolicacids), polyhydroxyalkanoates such as poly3-hydroxybutyrate orpoly4-hydroxybutyrate; polycaprolactones; poly(orthoesters);polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones);poly(glycolide-co-caprolactones); polycarbonates such as tyrosinepolycarbonates; polyamides (including synthetic and natural polyamides),polypeptides, and poly(amino acids); polyesteramides; otherbiocompatible polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophilic polyethers; polyurethanes; polyetheresters; polyacetals;polycyanoacrylates; polysiloxanes; poly(oxyethylene)/poly(oxypropylene)copolymers; polyketals; polyphosphates; polyhydroxyvalerates;polyalkylene oxalates; polyalkylene succinates; poly(maleic acids),polyvinyl alcohols, polyvinylpyrrolidone; poly(alkylene oxides) such aspolyethylene glycol (PEG); derivativized celluloses such as alkylcelluloses (e.g., methyl cellulose), hydroxyalkyl celluloses (e.g.,hydroxypropyl cellulose), cellulose ethers, cellulose esters,nitrocelluloses, polymers of acrylic acid, methacrylic acid orcopolymers or derivatives thereof including esters, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate) (jointly referred to herein as“polyacrylic acids”), as well as derivatives, copolymers, and blendsthereof.

As used herein, “derivatives” include polymers having substitutions,additions of chemical groups and other modifications to the polymericbackbones described above routinely made by those skilled in the art.Natural polymers, including proteins such as albumin, collagen, gelatin,prolamines, such as zein, and polysaccharides such as alginate andpectin, may also be incorporated into the polymeric matrix. While avariety of polymers may be used to form the polymeric matrix, generally,the resulting polymeric matrix will be a hydrogel. In certain cases,when the polymeric matrix contains a natural polymer, the naturalpolymer is a biopolymer which degrades by hydrolysis, such as apolyhydroxyalkanoate.

The polymeric matrix may optionally contain one or more crosslinkablepolymers. Preferably, the crosslinkable polymers contain one or morephoto-polymerizable groups, allowing for the crosslinking of thepolymeric matrix following nanolipogel formation. Examples of suitablephoto-polymerizable groups include vinyl groups, acrylate groups,methacrylate groups, and acrylamide groups. Photo-polymerizable groups,when present, may be incorporated within the backbone of thecrosslinkable polymers, within one or more of the sidechains of thecrosslinkable polymers, at one or more of the ends of the crosslinkablepolymers, or combinations thereof.

The polymeric matrix may be formed from polymers having a variety ofmolecular weights, so as to form nanolipogels having properties,including drug release rates, optimal for specific applications.Generally, the polymers which make up the polymeric matrix possessaverage molecular weights ranging between about 500 Da and 50 kDa. Incases where the polymeric matrix is formed from non-crosslinkablepolymers, the polymers typically possess average molecular weightsranging between about 1 kDa and about 50 kDa, more preferably betweenabout 1 kDa and about 70 kDa, most preferably between about 5 kDa andabout 50 kDa. In cases where the polymeric matrix is formed fromcrosslinkable polymers, the polymers typically possess lower averagemolecular weights ranging between about 500 Da and about 25 kDa, morepreferably between about 1 kDa and about 10 kDa, most preferably betweenabout 3 kDa and about 6 kDa. In particular embodiments the polymericmatrix is formed from a crosslinkable polymer having an averagemolecular weight of about 5 kDa.

In some embodiments, the polymeric matrix is formed from a poly(alkyleneoxide) polymer or a block copolymer containing one or more poly(alkyleneoxide) segments. The poly(alkylene oxide) polymer or poly(alkyleneoxide) polymer segments may contain between 8 and 500 repeat units, morepreferably between 40 and 300 repeat units, most preferably between 50and 150 repeat units. Suitable poly(alkylene oxides) includepolyethylene glycol (also referred to as polyethylene oxide or PEG),polypropylene 1,2-glycol, poly(propylene oxide), polypropylene1,3-glycol, and copolymers thereof.

In some embodiments, the polymeric matrix is formed from an aliphaticpolyester or a block copolymer containing one or more aliphaticpolyester segments. Preferably the polyester or polyester segments arepoly(lactic acid) (PLA), poly(glycolic acid) (PGA), orpoly(lactide-co-glycolide) (PLGA).

In some embodiments, the polymeric matrix is formed from a blockcopolymer containing one or more poly(alkylene oxide) segments, one ormore aliphatic polyester segments, and optionally one or morephoto-polymerizable groups. In these cases, the one or morepoly(alkylene oxide) segments imbue the polymer with the necessaryhydrophilicity, such that the resultant polymer matrix forms a suitablehydrogel, while the polyester segments provide a polymeric matrix withtunable hydrophobicity/hydrophilicity and/or the desired in vivodegradation characteristics.

The degradation rate of the polyester segments, and often thecorresponding drug release rate, can be varied from days (in the case ofpure PGA) to months (in the case of pure PLA), and may be readilymanipulated by varying the ratio of PLA to PGA in the polyestersegments. In addition, the poly(alkylene oxides), such as PEG, andaliphatic polyesters, such as PGA, PLA, and PLGA have been establishedas safe for use in humans; these materials have been used in humanclinical applications, including drug delivery systems, for more than 30years.

In certain embodiments, the polymeric matrix is formed from a tri-blockcopolymer containing a central poly(alkylene oxide) segment, adjoiningaliphatic polyester segments attached to either end of the centralpoly(alkylene oxide) segment, and one or more photo-polymerizablegroups. Preferably, the central poly(alkylene oxide) segment is PEG, andaliphatic polyesters segments are PGA, PLA, or PLGA.

Generally, the average molecular weight of the central poly(alkyleneoxide) segment is greater than the average molecular weight of theadjoining polyester segments. In certain embodiments, the averagemolecular weight of the central poly(alkylene oxide) segment is at leastthree times greater than the average molecular weight of one of theadjoining polyester segments, more preferably at least five timesgreater than the average molecular weight of one of the adjoiningpolyester segments, most preferably at least ten times greater than theaverage molecular weight of one of the adjoining polyester segments.

In some cases, the central poly(alkylene oxide) segment possesses anaverage molecular weight ranging between about 500 Da and about 10,000Da, more preferably between about 1,000 Da and about 7,000 Da, mostpreferably between about 2,500 Da and about 5,000 Da. In particularembodiments, average molecular weight of the central poly(alkyleneoxide) segment is about 4,000 Da. Typically, each adjoining polyestersegment possesses an average molecular weight ranging between about 100Da and about 3,500 Da, more preferably between about 100 Da and about1,000 Da, most preferably between about 100 Da and about 500 Da.

Examples of natural polymers include proteins such as albumin, collagen,gelatin and prolamines, for example, zein, and polysaccharides such asalginate, cellulose derivatives and polyhydroxyalkanoates, for example,polyhydroxybutyrate. The in vivo stability of the microparticles can beadjusted during the production by using polymers such aspoly(lactide-co-glycolide) copolymerized with polyethylene glycol (PEG).If PEG is exposed on the external surface, it may increase the timethese materials circulate due to the hydrophilicity of PEG.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

The matrix can also be made of gel-type polymers, such as alginate,produced through traditional ionic gelation techniques. The polymers arefirst dissolved in an aqueous solution, mixed with barium sulfate orsome bioactive agent, and then extruded through a microdroplet formingdevice, which in some instances employs a flow of nitrogen gas to breakoff the droplet. A slowly stirred (approximately 100-170 RPM) ionichardening bath is positioned below the extruding device to catch theforming microdroplets. The microparticles are left to incubate in thebath for twenty to thirty minutes in order to allow sufficient time forgelation to occur. Microparticle size is controlled by using varioussize extruders or varying either the nitrogen gas or polymer solutionflow rates. Chitosan microparticles can be prepared by dissolving thepolymer in acidic solution and crosslinking it with tripolyphosphate.Carboxymethyl cellulose (CMC) microparticles can be prepared bydissolving the polymer in acid solution and precipitating themicroparticle with lead ions. In the case of negatively charged polymers(e.g., alginate, CMC), positively charged ligands (e.g., polylysine,polyethyleneimine) of different molecular weights can be ionicallyattached.

Perhaps the most widely used are the aliphatic polyesters, specificallythe hydrophobic poly(lactic acid) (PLA), more hydrophilic PGA and theircopolymers, poly(lactide-co-glycolide) (PLGA). The degradation rate ofthese polymers, and often the corresponding drug release rate, can varyfrom days (PGA) to months (PLA) and is easily manipulated by varying theratio of PLA to PGA. Second, the physiologic compatibility of PLGA andits homopolymers PGA and PLA have been established for safe use inhumans; these materials have a history of over 30 years in various humanclinical applications including drug delivery systems. PLGAnanoparticles can be formulated in a variety of ways that improve drugpharmacokinetics and biodistribution to target tissue by either passiveor active targeting. The microparticles are designed to releasemolecules to be encapsulated or attached over a period of days to weeks.Factors that affect the duration of release include pH of thesurrounding medium (higher rate of release at pH 5 and below due to acidcatalyzed hydrolysis of PLGA) and polymer composition. Aliphaticpolyesters differ in hydrophobicity and that in turn affects thedegradation rate. Specifically the hydrophobic poly(lactic acid) (PLA),more hydrophilic PGA and their copolymers, poly(lactide-co-glycolide)(PLGA) have various release rates. The degradation rate of thesepolymers, and often the corresponding drug release rate, can vary fromdays (PGA) to months (PLA) and is easily manipulated by varying theratio of PLA to PGA.

Shell Components. Nanolipogels include a liposomal shell composed of oneor more concentric lipid monolayers or lipid bilayers. The shell canfurther include one or more active agents, targeting molecules, orcombinations thereof.

Nanolipogels include a liposomal shell composed of one or moreconcentric lipid monolayers or lipid bilayers. The composition of theliposomal shell may be varied to influence the release rate of one ormore active agents in vivo. The lipids may also be covalentlycrosslinked, if desired, to alter in vivo drug release.

The lipid shell can be formed from a single lipid bilayer (unilamellar)or several concentric lipid bilayers (multilamellar). The lipid shellmay be formed from a single lipid; however, in preferred embodiments,the lipid shell is formed from a combination of more than one lipid. Thelipids can be neutral, anionic, or cationic at physiologic pH.

Suitable neutral and anionic lipids include sterols and lipids such ascholesterol, phospholipids, lysolipids, lysophospholipids, andsphingolipids. Neutral and anionic lipids include, but are not limitedto, phosphatidylcholine (PC) (such as egg PC, soy PC), including1,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS),phosphatidylglycerol, phosphatidylinositol (PI); glycolipids;sphingophospholipids, such as sphingomyelin; sphingoglycolipids (alsoknown as 1-ceramidyl glucosides), such as ceramide galactopyranoside,gangliosides and cerebrosides; fatty acids, sterols containing acarboxylic acid group such as cholesterol or derivatives thereof; and1,2-diacyl-sn-glycero-3-phosphoethanolamines, including1,2-dioleoyl-sn-Glycero-3-phosphoethanolamine or 1,2-dioleolylglycerylphosphatidylethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine(DHPE), 1,2-distearoylphosphatidylcholine (DSPC),1,2-dipalmitoylphosphatidylcholine (DPPC), and1,2-dimyristoylphosphatidylcholine (DMPC). Also suitable are natural(e.g., tissue derived L-.alpha.-phosphatidyl: egg yolk, heart, brain,liver, soybean) and/or synthetic (e.g., saturated and unsaturated1,2-diacyl-sn-glycero-3-phosphocholines,1-acyl-2-acyl-sn-glycero-3-phosphocholines,1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of theselipids.

Suitable cationic lipids includeN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, alsoreferred to as TAP lipids, for example as a methylsulfate salt. SuitableTAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP(dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Othersuitable cationic lipids include dimethyldioctadecyl ammonium bromide(DDAB), 1,2-diacyloxy-3-trimethylammonium propanes,N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP),1,2-diacyloxy-3-dimethylammonium propanes,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1,2-dialkyloxy-3-dimethylammonium propanes,dioctadecylamidoglycylspermine (DOGS),3-[N—(N′,N′-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol);2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanam-iniumtrifluoro-acetate (DOSPA), .beta.-alanyl cholesterol,cetyltrimethylammonium bromide (CTAB), diC₁₄-amidine,N-tert-butyl-N′-tetradecyl-3-tetradecylamino-propionamidine,N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG),ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride,1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), andN,N,N′,N′-tetramethyl-,N′-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide,1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride derivatives, such as1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)-imidazoliniumchloride (DOTIM) and1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoliniumchloride (DPTIM), and 2,3-dialkyloxypropyl quaternary ammoniumderivatives containing a hydroxyalkyl moiety on the quaternary amine,for example, 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide(DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide(DORIE-HP), 1,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammoniumbromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe),1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide(DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE).

Other suitable lipids include PEGylated derivatives of the neutral,anionic, and cationic lipids described above. Incorporation of one ormore PEGylated lipid derivatives into the lipid shell can result in ananolipogel which displays polyethylene glycol chains on its surface.The resulting nanolipogels may possess increased stability andcirculation time in vivo as compared to nanolipogels lacking PEG chainson their surfaces. Examples of suitable PEGylated lipids includedistearoylphosphatidylethanlamine-polyethylene glycol (DSPE-PEG),including DSPE PEG (2000 MVW) and DSPE PEG (5000 MVW),dipalmitoyl-glycero-succinate polyethylene glycol (DPGS-PEG),stearyl-polyethylene glycol and cholesteryl-polyethylene glycol.

In certain embodiments, the lipid shell is formed from a combination ofmore than one lipid. In certain embodiments the lipid shell is formedfrom a mixture of at least three lipids. In particular embodiments, thelipid shell is formed from a mixture of phosphatidyl choline (PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol.

In some embodiments, the lipid shell is formed from a mixture of one ormore PEGylated phospholipids and one or more additional lipids orsterols. In certain instances, the molar ratio of the one or morePEGylated lipids to the one or more additional lipids or sterols rangesfrom about 1:1 to about 1:6, more preferably from about 1:2 to about1:6, most preferably from about 1:3 to about 1:5. In particularembodiments, the molar ratio of the one or more PEGylated lipids to theone or more additional lipids or sterols is about 1:4.

In some embodiments, the lipid shell is formed from a mixture of one ormore phospholipids and one or more additional lipids or sterols. Incertain instances, the molar ratio of the one or more phospholipids tothe one or more additional lipids or sterols ranges from about 1:1 toabout 6:1, more preferably from about 2:1 to about 6:1, most preferablyfrom about 3:1 to about 5:1. In particular embodiments, the molar ratioof the one or more phospholipids to the one or more additional lipids orsterols is about 4:1.

In preferred embodiments, the lipid shell is formed from a mixture of aphospholipid, such as phosphatidyl choline (PC), a PEGylatedphospholipid, such as1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol. In particular embodiments,the lipid shell is formed from a mixture of phosphatidyl choline,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol in a 3:1:1 molar ratio.

Polymeric Particles. The delivery vehicle can also be a polymericparticle, for example a micro- or a nanoparticle. The particles can bebiodegradable or non-biodegradable. Exemplary polymers that can be usedto manufacture polymeric particles are discussed above with respect tothe polymeric matrix component of nanolipogels.

Examples of preferred biodegradable polymers include polymers of hydroxyacids such as lactic acid and glycolic acid, and copolymers with PEG,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid),poly(valeric acid), poly(lactide-co-caprolactone), blends and copolymersthereof. In preferred embodiments, the particles are composed of one ormore polyesters.

For example, particles can contain one or more of the followingpolyesters: homopolymers including glycolic acid units, referred toherein as “PGA”, and lactic acid units, such as poly-L-lactic acid,poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide,poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as“PLA”, and caprolactone units, such as poly(.epsilon.-caprolactone),collectively referred to herein as “PCL”; and copolymers includinglactic acid and glycolic acid units, such as various forms ofpoly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide)characterized by the ratio of lactic acid:glycolic acid, collectivelyreferred to herein as “PLGA”; and polyacrylates, and derivativesthereof. Exemplary polymers also include copolymers of polyethyleneglycol (PEG) and the aforementioned polyesters, such as various forms ofPLGA-PEG or PLA-PEG copolymers, collectively referred to herein as“PEGylated polymers”. In certain embodiments, the PEG region can becovalently associated with polymer to yield “PEGylated polymers” by acleavable linker. Alginate polymers may also be used.

In some embodiments, the particles are composed of PLGA. PLGA is a safe,FDA approved polymer. PLGA particles are advantageous because they canprotect the active agent (i.e., the encapsulant), promote prolongedrelease, and are amenable to the addition of targeting moieties.

The particles can contain one or more polymer conjugates containingend-to-end linkages between the polymer and a targeting moiety,detectable label, or other active agent. For example, a modified polymercan be a PLGA-PEG-phosphonate. In another example, the particle ismodified to include an avidin moiety and a biotinylated targetingmoiety, detectable label, or other active agent can be coupled thereto.

Examples of preferred natural polymers include proteins such as albumin,collagen, gelatin and prolamines, for example, zein, and polysaccharidessuch as alginate, cellulose derivatives and polyhydroxyalkanoates, forexample, polyhydroxybutyrate. The in vivo stability of the particles canbe adjusted during the production by using polymers such aspoly(lactide-co-glycolide) copolymerized with polyethylene glycol (PEG).If PEG is exposed on the external surface, it may increase the timethese materials circulate due to the hydrophilicity of PEG.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Nanolipogels. A nanolipogel is a nanoparticle that combines theadvantages of both liposomes and polymer-based particles for sustaineddelivery of nucleic acids, proteins and/or small molecules. Thenanolipogel can be in the form of spheres, discs, rods or othergeometries with different aspect ratios. The nanosphere can be larger,i.e., microparticles. The nanolipogel is typically formed of syntheticor natural polymers capable of encapsulating agents by remote loadingand tunable in properties so as to facilitate different rates ofrelease. Release rates are modulated by varying the polymer to lipidratio from 0.05 to 5.0, more preferably from 0.5 to 1.5.

Nanolipogels are designed to be loaded with agents either prior to,during or after formation and subsequently function ascontrolled-release vehicles for the agents. The nanolipogel can beloaded with more than one agent such that controlled release of themultiplicity of agents is subsequently achieved.

The nanolipogel is loaded with at least one mitotic kinase inhibitor andat least one immune checkpoint inhibitor during formation and/orfollowing formation by the process of rehydration of the nanolipogel inthe presence of the agents. For example, the nanolipogel is loaded witha molecule that serves as a mitotic kinase inhibitor and the nanolipogelthereafter incorporates one or more immune checkpoint inhibitor afterformation (or vice versa), for the co-delivery and release of bothinhibitors together.

Polymeric Nanoparticles

Emulsion Method. In some embodiments, the polymeric nanoparticle isprepared using an emulsion solvent evaporation method. For example, apolymeric material is dissolved in a water immiscible organic solventand mixed with a drug solution or a combination of drug solutions. Thewater immiscible organic solvent can be, but is not limited to, one ormore of the following: chloroform, dichloromethane, and acyl acetate.The drug can be dissolved in, but is not limited to, one or more of thefollowing: acetone, ethanol, methanol, isopropyl alcohol, acetonitrileand dimethyl sulfoxide (DMSO). An aqueous solution is then added intothe resulting mixture solution to yield emulsion solution byemulsification. The emulsification technique can be, but is not limitedto, probe sonication or homogenization through a homogenizer. Thepeptides or fluorophores or drugs may be associated with the surface of,encapsulated within, surrounded by, and/or distributed throughout, thepolymeric matrix of the particle.

Nanoprecipitation Method. In another embodiment, the polymericnanoparticles are prepared using nanoprecipitation methods ormicrofluidic devices. A polymeric material is mixed with a drug or drugcombinations in a water miscible organic solvent. The resulting mixturesolution is then added to an aqueous solution to yield a nanoparticlesolution.

Exemplary Methods of Preparation. Particles can be fabricated fromdifferent polymers using a variety of methods that can be selected basedon criteria including the polymeric composition of the particle, theagent(s) being loaded into or associated with the particle according tomethod that are known in the art. Exemplary methods are provided below.

Solvent Evaporation. In this method the polymer is dissolved in avolatile organic solvent, such as methylene chloride. The drug (eithersoluble or dispersed as fine particles) is added to the solution, andthe mixture is suspended in an aqueous solution that contains a surfaceactive agent such as poly(vinyl alcohol). The resulting emulsion isstirred until most of the organic solvent evaporated, leaving solidparticles. The resulting particles are washed with water and driedovernight in a lyophilizer. Particles with different sizes (0.5-1000microns) and morphologies can be obtained by this method. This method isuseful for relatively stable polymers like polyesters and polystyrene.

However, labile polymers, such as polyanhydrides, may degrade during thefabrication process due to the presence of water. For these polymers,the following two methods, which are performed in completely anhydrousorganic solvents, are more useful.

Hot Melt Microencapsulation. In this method, the polymer is first meltedand then mixed with the solid particles. The mixture is suspended in anon-miscible solvent (like silicon oil), and, with continuous stirring,heated to 5° C. above the melting point of the polymer. Once theemulsion is stabilized, it is cooled until the polymer particlessolidify. The resulting particles are washed by decantation withpetroleum ether to give a free-flowing powder. Particles with sizesbetween 0.5 to 1000 microns are obtained with this method. The externalsurfaces of spheres prepared with this technique are usually smooth anddense. This procedure is used to prepare particles made of polyestersand polyanhydrides. However, this method is limited to polymers withmolecular weights between 1,000-50,000.

Solvent Removal. This technique is primarily designed forpolyanhydrides. In this method, the drug is dispersed or dissolved in asolution of the selected polymer in a volatile organic solvent likemethylene chloride. This mixture is suspended by stirring in an organicoil (such as silicon oil) to form an emulsion. Unlike solventevaporation, this method can be used to make particles from polymerswith high melting points and different molecular weights. Particles thatrange between 1-300 microns can be obtained by this procedure. Theexternal morphology of spheres produced with this technique is highlydependent on the type of polymer used.

Spray-Drying. In this method, the polymer is dissolved in organicsolvent. A known amount of the active drug is suspended (insolubledrugs) or co-dissolved (soluble drugs) in the polymer solution. Thesolution or the dispersion is then spray-dried. Typical processparameters for a mini-spray drier (Buchi) are as follows: polymerconcentration=0.04 g/mL, inlet temperature=−24° C., outlettemperature=13-15° C., aspirator setting=15, pump setting=10 mL/minute,spray flow=600 NI/hr, and nozzle diameter=0.5 mm. Microparticles rangingbetween 1-10 microns are obtained with a morphology which depends on thetype of polymer used.

Hydrogel Particles. Particles made of gel-type polymers, such asalginate, are produced through traditional ionic gelation techniques.The polymers are first dissolved in an aqueous solution, mixed withbarium sulfate or some bioactive agent, and then extruded through amicrodroplet forming device, which in some instances employs a flow ofnitrogen gas to break off the droplet. A slowly stirred (approximately100-170 RPM) ionic hardening bath is positioned below the extrudingdevice to catch the forming microdroplets. The particles are left toincubate in the bath for twenty to thirty minutes in order to allowsufficient time for gelation to occur. Particle size is controlled byusing various size extruders or varying either the nitrogen gas orpolymer solution flow rates. Chitosan particles can be prepared bydissolving the polymer in acidic solution and crosslinking it withtripolyphosphate. Carboxymethyl cellulose (CMC) particles can beprepared by dissolving the polymer in acid solution and precipitatingthe particle with lead ions. In the case of negatively charged polymers(e.g., alginate, CMC), positively charged ligands (e.g., polylysine,polyethyleneimine) of different molecular weights can be ionicallyattached.

Other Delivery Vehicles

In some embodiments, the delivery vehicles are liposomes or lipidnanoparticles. Liposomes are typically spherical vesicles composed of alamellar phase lipid bilayer. The liposomes can be, for example,multilamellar vesicles (MLV), small unilamellar liposome vesicles (SUV),large unilamellar vesicles (LUV), or cochleate vesicles. Liposomes,micelles, and other lipid-based delivery vehicles useful for preparationof the disclosed nanoparticulate compositions are known in the art. See,for example, Torchilin et al. (Adv Drug Delivery Rev, 58(14):1532-55,2006). It is anticipated that a wide variety of liposomes and exosomesmay be used with the present invention. Liposomes may compriseN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP) or Lipofectamine™ In some embodiments, a delivery systeminvolving chitosan may be used as described, e.g., in Lu et al. (CancerCell, 18:185-197, 2010). In some embodiments, a nanovector may be usedto deliver a miRNA to a subject; nanovectors are described, e.g., inPramanik et al. (Mol Cancer Ther, 10:1470-1480, 2011).

The delivery vehicle can also be silica particles. Suitable silicaparticles useful for preparation of the disclosed nanoparticulatecompositions are also known in the art. See, for example, Barbe et al.(Adv Materials, 16(21):1959-1966, 2004), Ngamcherdtrakul et al. (AdvFunc Materials, 25: 2646-2659, 2015) and Argyo et al. (Chem. Mater.,26(1):435-451, 2014). For example, in some embodiments, a siliconenanoparticle (e.g., as described in Bharali et al. PNAS, 102(32):11539-11544, 2005) may be used to deliver at least one mitotic kinaseinhibitor and at least one immune checkpoint inhibitor to a cell.Solubility of silica or silicon in the body provides the ability fortime-release of the agents that the particles carry. In addition,biodegradable polymers or bioreducible crosslinking agents can be usedto modify the silica or silicon particles to provide the time-releaseability.

(VI) ANTIBODIES

At least some of the agents herein (such as agents used to induce immunecheckpoint blockade) are antibodies. An antibody is a type of bindingagent, which is a molecule that can bind a target ligand, for instanceon the surface of a cell or in a biological sample. The term antibodyincludes both whole antibodies and functional (that is, maintainingsignificant and specific target binding) fragments thereof. The terms“antibody” and “immunoglobulin” are used interchangeably herein and arewell understood by those in the field. Those terms refer to a proteinincluding one or more polypeptides that specifically binds an antigen.One form of antibody includes the basic structural unit of an antibody.This form is a tetramer and includes two pairs of antibody chains, eachpair having one light and one heavy chain. In each pair, the light andheavy chain variable regions are together responsible for binding to theantigen recognized by that antibody, and the constant regions areresponsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of 25 kDa or 214 amino acids) include avariable region of 110 amino acids at the NH₂-terminus and a kappa orlambda constant region at the COOH-terminus. Full-length immunoglobulin“heavy chains” (of 50 kDa or 446 amino acids), similarly include avariable region (of 116 amino acids) and one of the aforementioned heavychain constant regions, e.g., gamma (of 330 amino acids).

Particular embodiments of antibodies and immunoglobulins includeantibodies or immunoglobulins of any isotype, fragments of antibodieswhich retain specific binding to antigen, including, Fab, Fv, scFv, andFd fragments, chimeric antibodies, humanized antibodies, single-chainantibodies, and fusion proteins including an antigen-binding portion ofan antibody and a non-antibody protein. The antibodies may be detectablylabeled, e.g., with a radioisotope, an enzyme which generates adetectable product, a fluorescent protein, a fluorescent molecule, or astable elemental isotope and the like. The antibodies may be furtherconjugated to other moieties, such as members of specific binding pairs,e.g., biotin (member of a biotin-avidin specific binding pair), and thelike. Also encompassed by the term are Fab′, Fv, F(ab′)₂, and otherantibody fragments that retain specific binding to their cognateantigen, and monoclonal antibodies.

Antibodies may exist in a variety of other forms including, for example,bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchiaet al., Eur. J. Immunol. 17: 105, 1987) and in single chains (e.g.,Huston et al., Proc. Natl. Acad. Sci. U.S.A. 85: 5879-5883, 1988; andBird et al., Science 242: 423-426, 1988). See, generally, Hood et al.(1984) “Immunology”, N.Y., 2nd ed., and Hunkapiller & Hood (Nature 323:15-16, 1986).

An immunoglobulin light or heavy chain variable region consists of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs has been precisely defined (see,“Sequences of Proteins of Immunological Interest” E. Kabat et al. (1991)US Department of Health and Human Services). In particular embodiments,the numbering of an antibody amino acid sequence can conform to theKabat system. The sequences of the framework regions of different lightor heavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs. The CDRs are primarily responsible for binding to an epitope of anantigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, from antibodyvariable and constant region genes belonging to different species. Forexample, the variable segments of the genes from a rabbit monoclonalantibody may be joined to human constant segments, such as γ1 and γ3.

(VII) PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION FORMULATIONS

Provided herein are compositions for use in treating cancer, precancer,and other proliferative disease. The compositions include at least twoactive components/agents, one of which is a therapeutically active agentthat inhibits at least one mitotic kinase inhibitor; and another ofwhich is an immune checkpoint inhibitor. As described herein, the activeagents may be delivered in/associated with a delivery vehicle (aconstruct, an engineered construct), such as a liposome, an organic orinorganic (nano- or micro-) particle, and so forth. As described herein,the active agents may be co-delivered with a chemical linker connectingthe agents (e.g., an antibody-drug conjugate, anantibody-oligonucleotide conjugate, a small molecule-oligonucleotideconjugate, or a small molecule-small molecule conjugate).

The compositions can be provided to the cells either directly, such asby contacting it with the cell, or indirectly, such as through theaction of any biological process. For example, the compositions can beformulated in a physiologically acceptable carrier or vehicle, andinjected into a tissue or fluid surrounding the cell. The compositionscan cross the cell membrane by simple diffusion, endocytosis, or by anyactive or passive transport mechanism.

When formulated in a pharmaceutical composition, a therapeutic compound(such as delivery system coupled with at least one mitotic kinaseinhibitor and at least one immune checkpoint inhibitor) can be admixedwith a pharmaceutically acceptable carrier or excipient. As used herein,the phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are generally believed to be physiologicallytolerable and do not typically produce an allergic or similar untowardreaction, such as gastric upset, dizziness and the like, whenadministered to a human or veterinary subject.

The term “pharmaceutically acceptable derivative” as used herein meansany pharmaceutically acceptable salt, solvate or prodrug, e.g. ester, ofthe desired active agent, which upon administration to the recipient iscapable of providing (directly or indirectly) the desired active agent,or an active metabolite or residue thereof. Such derivatives arerecognizable to those skilled in the art, without undue experimentation.Nevertheless, reference is made to the teaching of Burger's MedicinalChemistry and Drug Discovery, 5th Edition, Vol 1: Principles andPractice. Pharmaceutically acceptable derivatives include salts,solvates, esters, carbamates, and phosphate esters.

While it is possible to use a composition for therapy as is, it may bepreferable to administer it in a pharmaceutical formulation, e.g., inadmixture with a suitable pharmaceutical excipient, diluent or carrierselected with regard to the intended route of administration andstandard pharmaceutical practice. Accordingly, in one aspect,pharmaceutical composition or formulation includes at least one activecomposition, or a pharmaceutically acceptable derivative thereof, inassociation with a pharmaceutically acceptable excipient, diluent and/orcarrier. The excipient, diluent and/or carrier is “acceptable” in thesense of being compatible with the other ingredient(s) of theformulation and not significantly deleterious to the recipient thereof.

Any composition formulation disclosed herein can advantageously includeany other pharmaceutically acceptable carriers which include those thatdo not produce significantly adverse, allergic, or other untowardreactions that outweigh the benefit of administration, whether forresearch, prophylactic and/or therapeutic treatments. Exemplarypharmaceutically acceptable excipients, diluents, and carriers fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington: The Science and Practice ofPharmacy. Lippincott Williams & Wilkins (A. R., Gennaro edit. 2005), andin Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990. Moreover, formulations can be prepared to meet sterility,pyrogenicity, general safety and purity standards as required by UnitedStates FDA Office of Biological Standards and/or other relevant foreignregulatory agencies. The pharmaceutical excipient(s), diluent(s), andcarrier(s) can be selected with regard to the intended route ofadministration and standard pharmaceutical practice.

Such pharmaceutical formulations may be presented for use in aconventional manner with the aid of one or more suitable excipients,diluents, and carriers. Pharmaceutically acceptable excipients assist ormake possible the formation of a dosage form for a bioactive materialand include diluents, binding agents, lubricants, glidants,disintegrants, coloring agents, and other ingredients. Preservatives,stabilizers, dyes and even flavoring agents may be provided in thepharmaceutical composition. Examples of preservatives include sodiumbenzoate, ascorbic acid and esters of p-hydroxybenzoic acid.Antioxidants and suspending agents may be also used. An excipient ispharmaceutically acceptable if, in addition to performing its desiredfunction, it is non-toxic, well tolerated upon ingestion, and does notinterfere with absorption of bioactive materials.

Exemplary generally used pharmaceutically acceptable carriers includeany and all bulking agents or fillers, solvents or co-solvents,dispersion media, coatings, surfactants, antioxidants (e.g., ascorbicacid, methionine, vitamin E), preservatives, isotonic agents, absorptiondelaying agents, salts, stabilizers, buffering agents, chelating agents(e.g., EDTA), gels, binders, disintegration agents, and/or lubricants.

Exemplary buffering agents include citrate buffers, succinate buffers,tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers,lactate buffers, acetate buffers, phosphate buffers, histidine buffersand/or trimethylamine salts.

Exemplary preservatives include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides, hexamethonium chloride, alkyl parabenssuch as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and3-pentanol.

Exemplary isotonic agents include polyhydric sugar alcohols includingtrihydric or higher sugar alcohols, such as glycerin, erythritol,arabitol, xylitol, sorbitol, or mannitol.

Exemplary stabilizers include organic sugars, polyhydric sugar alcohols,polyethylene glycol; sulfur-containing reducing agents, amino acids, lowmolecular weight polypeptides, proteins, immunoglobulins, hydrophilicpolymers, or polysaccharides.

A “therapeutically effective amount” or “therapeutically effective dose”means the amount of a compound that, when administered to a subject fortreating a state, disorder or condition, is sufficient to effect suchstate, disorder, or condition. The “therapeutically effective amount”will vary depending on the compound, the disease and its severity andthe age, weight, physical condition and responsiveness of the mammal tobe treated. The exact dose and formulation will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Remington: The Science and Practiceof Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, DosageCalculations (1999)). In certain cases, “therapeutically effectiveamount” is used to mean an amount or dose sufficient to modulate, e.g.,increase or decrease a desired activity e.g., by 10%, by 50%, or by 90%.Generally, a therapeutically effective amount is sufficient to cause animprovement in a clinically significant condition in the host followinga therapeutic regimen involving one or more therapeutic agents. Theconcentration or amount of the active ingredient depends on the desireddosage and administration regimen, as discussed herein.

The actual dose amount administered to a particular subject can bedetermined by a physician, veterinarian, or researcher taking intoaccount parameters such as physical, physiological and psychologicalfactors including target, body weight, stage of cancer, the type ofcancer, previous or concurrent therapeutic interventions, idiopathy ofthe subject, and route of administration.

Amounts effective for this use will depend on the severity of thedisease and its location, particularly when a metastatic site isimplicated, and the weight and general state of the patient beingtreated. Generally dosages range from 0.01 mg/kg to 100 mg/kg host bodyweight of therapeutic construct per day, with dosages of from 0.1 mg/kgto 10 mg/kg per day being more commonly used, and for instance dosagesof 3-7 mg/kg. Maintenance dosages over a prolonged period of time may beadjusted as necessary. The dosages, however, may be varied dependingupon the requirements of the patient, the severity of the conditionbeing treated, and the compound being employed. For example, dosages canbe empirically determined considering the type and stage of cancerdiagnosed in a particular patient. The dose administered to a patient,in the context of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects that accompany the administration of aparticular vector, or transduced cell type in a particular patient.Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day, if desired.

The selected dosage may be influenced by the desired therapeutic effect,the route of administration, the duration of the treatment desired, andthe specific therapeutic complex being employed. Generally, therapeuticconstruct can be administered in a range of about 0.001 mg/kg to 100mg/kg per administration (e.g., daily; or 2, 3, 4, 5 or more timesweekly; or 2, 3, 4, 5 or more times a month, etc., as discussed in moredetail below). The route of administration can be a consideration indetermining dosage as well. For example, in a particular embodiment, atherapeutic construct is administered in a range of 0.01 mg/kg to 100mg/kg (e.g., daily; or 2, 3, 4, 5 or more times weekly; or 2, 3, 4, 5 ormore times a month, etc.) by intravenous or interpretational routes, orin a range of 0.0001 mg/kg to 1 mg/kg (e.g., daily; or 2, 3, 4, 5 ormore times weekly; or 2, 3, 4, 5 or more times a month, etc.) for asubcutaneous route (e.g., local injection into or adjacent to a tumor orinto the TME). More exemplary dosage are discussed below.

Suitable dosages may range from 0.01 mg/kg to 100 mg/kg of body weightper day, week, or month. Exemplary doses can include 0.05 mg/kg to 10.0mg/kg of the active compounds (therapeutic constructs) disclosed herein.The total daily dose can be 0.05 mg/kg to 30.0 mg/kg of an agentadministered to a subject one to three times a day, includingadministration of total daily doses of 0.05-3.0, 0.1-3.0, 0.5-3.0,1.0-3.0, 1.5-3.0, 2.0-3.0, 2.5-3.0, and 0.5-3.0 mg/kg/day ofadministration forms of a drug using 60-minute oral, intravenous orother dosing. In one particular example, doses can be administered QD orBID to a subject with, e.g., total daily doses of 1.5 mg/kg, 3.0 mg/kg,4.0 mg/kg, 5.0 mg/kg, or 7.5 mg/kg of a composition with up to 92-98%wt/v of the compounds disclosed herein.

Additional useful doses can often range from 0.1 to 5 μg/kg or from 0.5to 1 μg/kg. In other examples, a dose can include 1 μg/kg, 10 μg/kg, 20μg/kg, 40 μg/kg, 80 μg/kg, 200 μg/kg, 0.1 to 5 mg/kg or from 0.5 to 1mg/kg. In other examples, a dose can include 1 mg/kg, 10 mg/kg, 20mg/kg, 40 mg/kg, 80 mg/kg, 200 mg/kg, 400 mg/kg, 450 mg/kg, or more.

Therapeutic materials of the present disclosure may be employed inserious disease states, that is, life-threatening or potentially lifethreatening situations. In such cases, it is possible and may be feltdesirable by the treating physician to administer substantial excessesof these compositions.

As will be appreciated by those of skill in the art, specific dosageswill be influenced by the pharmacokinetics of the active compound. Foradministration, therapeutically effective amounts (also referred toherein as doses) can be initially estimated based on results from invitro assays and/or animal model studies. Such information can be usedto more accurately determine useful doses in subjects of interest.Useful pre-clinical tests include pharmacodynamic analyses, toxicityanalyses, and so forth.

Therapeutically effective amounts can be achieved by administeringsingle or multiple doses during the course of a treatment regimen (e.g.,hourly, every 2 hours, every 3 hours, every 4 hours, every 6 hours,every 9 hours, every 12 hours, every 18 hours, daily, every other day,every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2weeks, every 3 weeks, or monthly).

The effective amounts of compounds containing active agents includedoses that partially or completely achieve the desired therapeutic,prophylactic, and/or biological effect. The actual amount effective fora particular application depends on the condition being treated and theroute of administration. The effective amount for use in humans can bedetermined from animal models. For example, a dose for humans can beformulated to achieve local (e.g., intratumoral) or circulating levelsthat have been found to be effective in animals.

Compositions can be administered with one or more anesthetics includingethanol, bupivacaine, chloroprocaine, levobupivacaine, lidocaine,mepivacaine, procaine, ropivacaine, tetracaine, desflurane, isoflurane,ketamine, propofol, sevoflurane, codeine, fentanyl, hydromorphone,marcaine, meperidine, methadone, morphine, oxycodone, remifentanil,sufentanil, butorphanol, nalbuphine, tramadol, benzocaine, dibucaine,ethyl chloride, xylocaine, and/or phenazopyridine.

In particular embodiments that include treating or preventing a cancer(including for instance a cancer metastasis), the compositions disclosedherein can be used in conjunction with other cancer treatments, such aschemotherapy, targeted therapy, radiation therapy, and/or immunotherapy.The compositions described herein can be administered simultaneouslywith or sequentially with another treatment within a selected timewindow, such as within 10 minutes, 1 hour, 3 hour, 10 hour, 15 hour, 24hour, or 48 hour time windows or when the complementary treatment iswithin a clinically-relevant therapeutic window.

Pharmaceutical compositions can be for administration by parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), by instillation, or in a depot, formulated in dosage formsappropriate for each route of administration.

In some embodiments, the compositions are administered systemically, forexample, by intravenous or intraperitoneal administration, in an amounteffective for delivery of the compositions to targeted cells. Otherroutes include instillation or mucosal.

In certain embodiments, the compositions are administered locally, forexample, by injection directly into a site to be treated. In someembodiments, the compositions are injected or otherwise administereddirectly to one or more tumors or diseased tissues. Typically, localinjection causes an increased localized concentration of thecompositions which is greater than that which can be achieved bysystemic administration. In some embodiments, the compositions aredelivered locally to the appropriate cells by using a catheter orsyringe. Other means of delivering such compositions locally to cellsinclude using infusion pumps or incorporating the compositions intopolymeric implants which can effect a sustained release of thecompositions to the immediate area of the implant.

By way of example, in certain embodiments the therapeutic constructs aregiven locally, for instance to readily accessible tumors such asmelanoma, head and neck cancer, breast cancer, and lymphoma; orsystemically for other cancers such as lung cancer, liver cancer,pancreatic cancer, prostate cancer, and metastatic cancers.

Thus, the therapeutic compositions described herein can be administered(on their own or as part of a combination therapy) by a variety ofroutes, including any convenient way for use in human or veterinarymedicine. A therapeutically effective amount of the desired activeagent(s) can be formulated in a pharmaceutical composition to beintroduced parenterally, transmucosally (e.g., orally, nasally, orrectally), or transdermally. In some embodiments, administration isparenteral, for instance, via intravenous injection, or intra-arteriole,intramuscular, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial administration. The administered maybe as a bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. In certain embodiments, for instance those involvedin treatment of inflammatory conditions that impact joints, thepharmaceutical composition may be administered directly to the synovium,synovial fluid or joint capsule by injection preferably with a syringe.Administration may be local or systemic; the choice may be influenced bythe condition being treated, as well as the active agent(s) andcompositions being administered.

For injection, compositions can be made as aqueous solutions, such as inbuffers such as Hanks' solution, Ringer's solution, or physiologicalsaline. The solutions can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the composition canbe in lyophilized and/or powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Compositions including a therapeutic construct may be administered in anaqueous solution, by parenteral injection. The injectable formulationcan be in the form of a suspension or emulsion, and optionally includespharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such injectable compositions caninclude diluents such as sterile water, buffered saline of variousbuffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionicstrength; and optionally, additives such as detergents and solubilizingagents (e.g., TWEEN™ 20, TWEEN™ 80 also referred to as polysorbate 20 or80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), andpreservatives (e.g., Thimerosal, benzyl alcohol). Examples ofnon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatin, andinjectable organic esters such as ethyl oleate. The formulations forinjection may be lyophilized and resuspended, for instance immediatelybefore use. The injectable formulation may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions.

In other embodiments, therapeutic construct-including compositions areapplied topically or by instillation. Topical administration can includeapplication to the lungs, nasal, oral (sublingual, buccal), vaginal, orrectal mucosa. These methods of administration can be made effective byformulating the shell or coating of the delivery vehicle with mucosaltransport element(s). Compositions can be delivered to the lungs whileinhaling and traverse across the lung epithelial lining to the bloodstream when delivered either as an aerosol or spray dried particleshaving an aerodynamic diameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator.

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations can includepenetration enhancers. Chemical enhancers and physical methods includingelectroporation and microneedles can work in conjunction with thismethod.

A microneedle (MN) is a micron-sized needle with a height of 10-2000 μmand a width of 10-50 μm, which can penetrate through the epidermis layerto dermal tissue directly with minimal or no pain (Hao et al., J BiomedNanotechnol, 13(12):1581-1597, 2017). Several types of microneedles canbe used. In some embodiments, metal-based or plastic microneedle rollerscan be used to physically disrupt skin surface to enhance penetration ofthe applied topical agents (therapeutic construct in this case). In someembodiments, degradable and dissolvable microneedles can containtherapeutic constructs. Upon administration to skin, microneedles candissolve and release the construct deep in layers of skin. In someembodiments, non-degradable microneedles may be coated with therapeuticconstructs, such that they deliver the coated construct deep in skinlayers. Microneedles can be fabricated from many classes of materials,including but not limited to, polymer, saccharides, polysaccharides,peptide, protein, metals, inorganic compound, and so forth (Ye et al.,Adv Drug Deliv Rev, 127: 106-118, 2018). All materials and fabricationmethods known in the art for microneedle technology is applicable toenhance delivery of this therapeutic construct.

Any device that facilitates systemic or localized delivery oftherapeutics is also applicable to the herein provided therapeuticconstructs. For example, hepatic arterial infusion (HAI) pump, which isan implanted chemotherapy device that delivers high concentrations ofcytotoxic agents directly to liver metastases with minimal systemictoxicities (Cohen et al., The Oncologist, 8(6): 553-566, 2003), can alsobe utilized to deliver the herein described therapeutic constructs. Insome embodiments, convection enhanced delivery (CED), which involves theplacement of a small diameter infusion catheter to deliver therapeuticsto brain tumors (Mehta, A. et al. Neurotherapeutics: the journal of theAmerican Society for Experimental Neuro Therapeutics, 14(2), 358-371,2017), can also be utilized to deliver the herein described therapeuticconstructs.

(VIII) EXEMPLARY METHODS OF USE

With the provision herein of therapeutic constructs that include atleast one mitotic kinase inhibitor and at least one immune checkpointinhibitor, there are now enabled methods of treating and/or preventinghyperproliferative diseases, disorders, or conditions, including cancer,symptoms of cancer, cancer progression (including from precancer tocancer), and cancer metastasis. Specific examples of hyperproliferativediseases, disorders, or conditions include cancer. In some embodiments,the cancer may suppress the immune system of the subject or individualwith the cancer. In some embodiments, the therapeutic constructs asprovided herein can suppress or reverse cancer-mediated immunesuppression and allow for immune recognition and clearance of themalignancy.

As used herein, the term “treatment” or “treating” refers to anyimprovement of the cancer that occurs in a treated subject compared toan untreated subject. Such an improvement can be a prevention of aworsening or progression of the cancer (e.g., improved progression-freesurvival). Moreover, such an improvement may also be a reduction or cureof the cancer or its accompanying symptoms (e.g., reduction in tumorvolume, partial remission, complete remission (e.g., for 6 months, 1year, 2 years, 3 years, 4 years, or 5 years or more), prevention ofcancer recurrence or relapse, reduction of metastasis, or reduction ofnumber of tumors or lesions). It will be understood that a treatment maynot be successful for 100% of the subjects to be treated. The term,however, requires that the treatment is successful as determined bypeople skilled in the art (e.g., oncologists, physicians). As usedherein, the term “preventing” refers to avoiding the onset of cancer asused herein or its accompanying syndromes. It will be understood thatprevention refers to avoiding the onset of cancer within a certain timewindow in the future. Said time window shall, preferably, start uponadministration of a compound in the sense of the invention and lasts forat least 1 month, at least 6 months, at least 9 months, at least 1 year,at least 2 years, at least 5 years, at least 10 years or even for theremaining physiological life span of a subject. It will be understoodthat a prevention may not be successful for 100% of the subjects to betreated. The term, however, requires that the prevention is successfulas determined by one skilled in the art (e.g., oncologists, physicians).Prevention may also be in the context of a recurrence of cancer afterremission, e.g., as measured by a reduction in probability forrecurrence in a population.

The disclosed compositions can be used to treat benign or malignantcancers, and tumors thereof. The treatment can directly target and killcancer cells, indirectly target the cancer cells by increasing an immuneresponse against the cancer cells; or a combination thereof.

In a mature animal, a balance usually is maintained between cell renewaland cell death in most organs and tissues. The various types of maturecells in the body have a given life span; as these cells die, new cellsare generated by the proliferation and differentiation of various typesof stem cells. Under normal circumstances, the production of new cellsis so regulated that the numbers of any particular type of cell remainconstant. Occasionally, though, cells arise that are no longerresponsive to normal growth-control mechanisms. These cells give rise toclones of cells that can expand to a considerable size, producing atumor or neoplasm. A tumor that is not capable of indefinite growth anddoes not invade the healthy surrounding tissue extensively is benign. Atumor that continues to grow and becomes progressively invasive ismalignant. The term cancer refers specifically to a malignant tumor. Inaddition to uncontrolled growth, malignant tumors exhibit metastasis. Inthis process, small clusters of cancerous cells dislodge from a tumor,invade the blood or lymphatic vessels, and are carried to other tissues,where they continue to proliferate. In this way a primary tumor at onesite can give rise to a secondary tumor at another site.

The disclosed compositions can delay or inhibit the growth of a tumor ina subject, reduce the growth or size of the tumor or eliminate italtogether, inhibit or reduce metastasis of the tumor, and/or inhibit orreduce symptoms associated with tumor development or growth. Forexample, in some embodiments, the compositions reduce tumor burden inthe subject or slow or prevent tumor growth over time.

Malignant tumors may be classified according to the embryonic origin ofthe tissue from which the tumor is derived. Carcinomas are tumorsarising from endodermal or ectodermal tissues such as skin or theepithelial lining of internal organs and glands. Sarcomas, which ariseless frequently, are derived from mesodermal connective tissues such asbone, fat, and cartilage. The leukemias and lymphomas are malignanttumors of hematopoietic cells of the bone marrow. Leukemias proliferateas single cells, whereas lymphomas tend to grow as tumor masses.Malignant tumors may show up at numerous organs or tissues of the bodyto establish a cancer.

The types of cancer that can be treated with the provided compositionsand methods include, but are not limited to, vascular cancers such asmultiple myeloma, as well as solid cancers, including adenocarcinomasand sarcomas, of bone, bladder, brain, breast, cervix, colon, rectum,esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin,stomach, and uterus. In some embodiments, the disclosed compositions areused to treat multiple cancer types concurrently. The compositions canalso be used to treat metastases or tumors at multiple locations.

Administration is not limited to the treatment of an existing tumor butcan also be used to prevent or lower the risk of developing suchdiseases in an individual, i.e., for prophylactic use and to reducespread of cancer, for instance through metastasis. Potential candidatesfor prophylactic vaccination include individuals with a high risk ofdeveloping cancer, i.e., with a personal or familial history of certaintypes of cancer.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals, including leukemia,lymphoma, carcinomas and sarcomas. Exemplary cancers that may be treatedwith a compound, pharmaceutical composition, or method provided hereininclude lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor,cervical cancer, colon cancer, esophageal cancer, gastric cancer, headand neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia,prostate cancer, breast cancer (e.g. triple negative, ER positive, ERnegative, chemotherapy resistant, Herceptin® resistant, HER2 positive,doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobularcarcinoma, primary, metastatic), ovarian cancer, pancreatic cancer,liver cancer (e.g. hepatocellular carcinoma), lung cancer (e.g.non-small cell lung carcinoma, squamous cell lung carcinoma,adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma,carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostatecancer, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g. head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma. Additional examples include, cancer of the thyroid,endocrine system, brain, breast, cervix, colon, head & neck, esophagus,liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary,sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma,glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, primary brain tumors, cancer,malignant pancreatic insulinoma, malignant carcinoid, urinary bladdercancer, premalignant skin lesions, testicular cancer, lymphomas, thyroidcancer, neuroblastoma, esophageal cancer, genitourinary tract cancer,malignant hypercalcemia, endometrial cancer, adrenal cortical cancer,neoplasms of the endocrine or exocrine pancreas, medullary thyroidcancer, medullary thyroid carcinoma, melanoma, colorectal cancer,papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease ofthe Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma,cancer of the pancreatic stellate cells, cancer of the hepatic stellatecells, or prostate cancer. The term “precancer”, as used herein, refersto a condition or growth that precedes or develops into a cancer. Theterm “cancer metastasis”, as used herein, refers to the spread of cancercells or a tumor from one organ or part of the body to another organ orpart of the body.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). Exemplary leukemias that may be treated with a compound,pharmaceutical composition, or method provided herein include, forexample, acute nonlymphocytic leukemia, chronic lymphocytic leukemia,acute granulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, or undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a compound, pharmaceuticalcomposition, or method provided herein include a chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcomaof B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, or telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas that may betreated with a compound, pharmaceutical composition, or method providedherein include, for example, acral-lentiginous melanoma, amelanoticmelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, nodular melanoma, subungal melanoma, or superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acompound, pharmaceutical composition, or method provided herein include,for example, medullary thyroid carcinoma, familial medullary thyroidcarcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma,adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenalcortex, alveolar carcinoma, alveolar cell carcinoma, basal cellcarcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamouscell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,bronchogenic carcinoma, cerebriform carcinoma, cholangiocellularcarcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma,corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinomacutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductcarcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lobularcarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinomavillosum.

(IX) KITS

Active component(s), including particularly at least one describedtherapeutic construct (including a delivery vehicle containing orassociated with at least one mitotic kinase inhibitor and at least oneimmune checkpoint inhibitor), can be provided as kits. Kits can includeone or more containers including (containing) one or more or morecompounds or complexes (e.g., anti-cancer agents) as described herein,optionally along with one or more additional agents for use in therapy.For instance, some kits will include an amount of at least oneadditional anti-cancer composition, or an amount of at least oneadditional anti-inflammatory agent, or both.

Any active component in a kit may be provided in premeasured dosages,though this is not required; and it is anticipated that certain kitswill include more than one dose.

Kits can also include a notice in the form prescribed by a governmentalagency regulating the manufacture, use, or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use, or sale for human administration. The notice may statethat the provided active ingredients can be administered to a subject.The kits can include further instructions for using the kit, forexample, instructions regarding administration; proper disposal ofrelated waste; and the like. The instructions can be in the form ofprinted instructions provided within the kit or the instructions can beprinted on a portion of the kit itself. Instructions may be in the formof a sheet, pamphlet, brochure, CD-ROM, or computer-readable device, orcan provide directions to instructions at a remote location, such as awebsite. In particular embodiments, kits can also include some or all ofthe necessary medical supplies needed to use the kit effectively, suchas applicators, ampules, sponges, sterile adhesive strips, Chloraprep,gloves, and the like. Variations in contents of any of the kitsdescribed herein can be made. The instructions of the kit will directuse of the active ingredient(s) included in that kit to effectuate aclinical and/or therapeutic use described herein.

Suitable methods, materials, and examples used in the practice and/ortesting of embodiments of the disclosed invention are described herein.Such methods and materials are illustrative only and are not intended tobe limiting. Other methods, materials, and examples similar orequivalent to those described herein can be used.

The Exemplary Embodiments and Example(s) below are included todemonstrate particular embodiments of the disclosure. Those of ordinaryskill in the art should recognize in light of the present disclosurethat many changes can be made to the specific embodiments disclosedherein and still obtain a like or similar result without departing fromthe spirit and scope of the disclosure.

(X) Exemplary Embodiments

1. A therapeutic construct including: a delivery system; at least onemitotic kinase inhibitor, e.g., coupled to or contained within thedelivery system; and at least one immune checkpoint inhibitor, e.g.,coupled to or contained within the delivery system.2. The therapeutic construct of embodiment 1, wherein the deliverysystem includes a liposome, a lipid-based particle, a polymericparticle, an inorganic or organic nanoparticle or microparticle, or ahybrid thereof.3. The therapeutic construct of embodiment 2, wherein the deliveryvehicle includes one or more of fullerenes, endohedralmetallofullerenes, trimetallic nitride templated endohedralmetallofullerenes, single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, microparticles, quantum dots, superparamagneticnanoparticles, calcium phosphate particles, aluminum salt particles,nanorods, cellulose nanoparticles, silicon, silica and polymer micro-and nano-spheres, silica-shells, biodegradable PLGA micro- andnano-spheres, gold nanoparticles, cerium oxide particles, zinc oxideparticles, silver nanoparticles, carbon nanoparticles, ironnanoparticles, and/or modified micelles.4. The therapeutic construct of any one of embodiments 1-3, wherein thedelivery vehicle comprises a mesoporous silica nanoparticle.5. The therapeutic construct of embodiment 4, wherein the mesoporoussilica nanoparticle has a size of about 5-200 nm.6. The therapeutic construct of embodiments 4 or 5, wherein themesoporous silica nanoparticle is coated with cross-linkedpolyethyleneimine and polyethylene glycol.7. The therapeutic construct of any one of embodiments 1-6, wherein themitotic kinase inhibitor and/or immune checkpoint inhibitor includes anoligonucleotide, a polynucleotide, a small molecule inhibitor, or anantibody.8. The therapeutic construct of any one of embodiments 1-7, wherein theat least one mitotic kinase inhibitor is an inhibitor of a polo-likekinase (PLK), an Aurora kinase, cyclin-dependent kinase (CDK)1, CDK2,HASPIN, monopolar spindle 1 kinase (Mps1), or a NimA-related kinase(NEK).9. The therapeutic construct of any one of embodiments 1-8, wherein themitotic kinase inhibitor includes one or more of GSK461364, BI2536,Tak960, NMS-P937, volasertib, Chk 1 Kinase Inhibitor LY2603618, AU14022,YK-4-279, AZ703, alisertib, prexasertib, or AZD7762.10. The therapeutic construct of any one of embodiments 1-9, wherein themitotic kinase inhibitor is volasertib.11. The therapeutic construct of any one of embodiments 1-10, whereinthe immune checkpoint inhibitor includes a siRNA, inhibitor, or antibodyagainst one or more of PD-L1, PD-1, TIM-3, LAG-3, or CTLA-4.12. The therapeutic construct of any one of embodiments 1-11, whereinthe at least one immune checkpoint inhibitor selected from an antibodyagainst PD-L1, PD-1, or CTLA-4.13. The therapeutic construct of any one of embodiments 1-12, whereinthe at least one immune checkpoint inhibitor is an antibody againstPD-L1.14. The therapeutic construct of embodiment 13, wherein the immunecheckpoint inhibitor includes at least one of: nivolumab, pembrolizumab,MPDL3280A, ipilimumab, tremelimumab, atezolizumab, avelumab, durvalumab,cemiplimab, pidilizumab, or spartalizumab.15. The therapeutic construct of any of the previous embodiments,further including an adjuvant.16. The therapeutic construct of embodiment 15, wherein the adjuvantincludes one or more of a CpG oligonucleotide, a DNA TLR agonistcontaining a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLRagonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, acytokine, a small molecule TLR agonist, an oil- or surfactant-basedadjuvant, a lipopolysaccharide, a plant extract, or a derivativethereof.17. The therapeutic construct of embodiment 15 or 16, wherein theadjuvant includes a CpG oligonucleotide, imiquimod, resiquimod,gardiquimod, poly I:C, poly ICLC, dSLIM, or EnanDIM.18. The therapeutic construct of embodiment 17, wherein the adjuvantcomprises a CpG oligonucleotide.19. The therapeutic construct of any one of embodiments 1-18, having ahydrodynamic size of 5-999 nm.20. The therapeutic construct of any one of embodiments 1-18, having ahydrodynamic size of 1-1000 microns.21. A therapeutic construct including: an immune checkpoint inhibitor; amitotic kinase inhibitor; and a chemical linker linking the immunecheckpoint inhibitor and the mitotic kinase inhibitor.22. The therapeutic construct of embodiment 21, wherein the mitotickinase inhibitor is an oligonucleotide, a polynucleotide, a smallmolecule inhibitor, or an antibody.23. The therapeutic construct of embodiment 21 or 22, wherein the immunecheckpoint inhibitor is an oligonucleotide, a polynucleotide, a smallmolecule inhibitor, or an antibody.24. The therapeutic construct of any one of embodiments 21-23, whereinthe immune checkpoint inhibitor is an antibody.25. The therapeutic construct of any one of embodiments 21-24, whereinthe immune checkpoint inhibitor is an antibody against PD-L1, PD-1,TIM-3, LAG-3, or CTLA-4.26. The therapeutic construct of any one of embodiments 21-25, whereinthe immune checkpoint inhibitor is an antibody against PD-L1, PD-1, orCTLA-4.27. The therapeutic construct of any one of embodiments 21-26, whereinthe immune checkpoint inhibitor is an antibody against PD-L1.28. The therapeutic construct of any one of embodiments 21-27, whereinthe mitotic kinase inhibitor is selected from GSK461364, BI2536, Tak960,NMS-P937, volasertib, Chk 1 Kinase Inhibitor LY2603618, AU14022,YK-4-279, AZ703, alisertib, prexasertib, or AZD7762.29. The therapeutic construct of any one of embodiments 21-28, whereinthe mitotic kinase inhibitor is alisertib.30. The therapeutic construct of any one of embodiments 21-29, whereinthe chemical linker comprises one or more of a hydrazine; a disulfide;N-succinimidyl-4-(2-pyridyldithio)butanoate;N-succinimidyl-4-(2-pyridyldithio)-2-sulfo butanoate; perfluorophenyl3-(pyridin-2-yldisulfanyl)propanoate; 2,5-dioxopyrrolidin-1-yl3-methyl-3-(pyridin-2-yldisulfanyl)butanoate; Gly-Phe-Leu-Gly;Ala-Leu-Ala-Leu; Val-Cit; Phe-Lys; Val-Ala; Ala-Phe-Lys; Phe-Lys;(Gly)_(n), wherein n is 1-20; a β-glucuronide linker; maleimidocaproyl;N-(maleimidomethyl)cyclohexane-1-carboxylate;4-(4-acetylphenoxy)butanoic acid; dibromomaleimide; para-aminobenzoicacid; 4-nitrophenol; acetic acid; formic acid; 4-maleimidobutyric acidN-succinimidyl ester; N-(4-maleimidobutyryloxy)succinimide;N-(6-maleimidocaproyloxy)succinimide; 3-maleimidopropionic acidN-succinimidyl ester; N-(3-maleimidopropionyloxy)succinimide;5-maleimidovalericacid-NHS; linear, branched, or multi-arm polyethyleneglycol having a molecular weight of 100-10000 Da;propargyl-N-hydroxysuccinimidyl ester; pyrophosphate;succimimidyl-4-azidobutyrate; 4-azidobenzoic acid N-hydroxysuccinimideester; tert-butyl1-(4-formylphenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-oate; or aresidue thereof.31. The therapeutic construct of any one of embodiments 21-30, whereinthe chemical linker comprisesN-(maleimidomethyl)cyclohexane-1-carboxylate linker or a residuethereof.32. The therapeutic construct of any one of embodiments 21-31, whereinthe chemical linker comprises sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate.33. The therapeutic construct of any one of embodiments 21-32, whereinthe chemical linker comprises a linear polyethyleneglycol having amolecular weight of 100-10000 Da, or a residue thereof.34. The therapeutic construct of any one of embodiments 21-33, whereinthe ratio of mitotic kinase inhibitor to immune checkpoint inhibitor isabout 1-20.35. The therapeutic construct of embodiment 34, wherein the ratio ofmitotic kinase inhibitor to immune checkpoint inhibitor is about 2-8.36. The therapeutic construct of embodiment 34 or 35, wherein the ratioof mitotic kinase inhibitor to immune checkpoint inhibitor is about 4-6.37. The therapeutic construct of any one of embodiments 21-33, whereinthe ratio of immune checkpoint inhibitor to mitotic kinase inhibitor isabout 1-20.38. The therapeutic construct of embodiment 37, wherein the ratio ofimmune checkpoint inhibitor to mitotic kinase inhibitor is about 2-8.39. The therapeutic construct of embodiment 37 or 38, wherein the ratioof immune checkpoint inhibitor to mitotic kinase inhibitor is about 4-6.40. A composition comprising the therapeutic construct of any one ofembodiments 1-39 and a pharmaceutically acceptable carrier, excipient,or diluent.41. A method of treating cancer comprising administering to a subjectwith cancer an effective amount of the therapeutic construct of any oneof embodiments 1-39, or the composition of embodiment 40.42. The method of embodiment 41, wherein the subject is a human.43. A method of treating a cell exhibiting symptoms of cancer comprisingcontacting the cell with a therapeutically effective amount of thetherapeutic construct of any one of embodiments 1-39, or a compositionof embodiment 40.44. A method of treating a cell obtained from a subject exhibitingsymptoms of cancer, comprising contacting the cell with atherapeutically effective amount of the therapeutic construct of any oneof embodiments 1-39, or the composition of embodiment 40.45. A method of treating a cell obtained from a subject exhibitingsymptoms of cancer, comprising contacting cell ex vivo with atherapeutically effective amount of the therapeutic construct of any oneof embodiments 1-39, or the composition of embodiment 40.46. The method of embodiment 44 or 45, wherein the cell is a cancercell.47. The method of embodiment 44 or 45, wherein the cell is not a cancercell.48. The method of embodiment 47, wherein the cell is an immune cell.49. The method of any one of embodiments 43-48, further comprisingadministering at least one treated cell back to a subject.50. A method of treating a subject diagnosed as having ahyperproliferative disease or condition, comprising administering to thesubject an effective amount of the composition of embodiment 40.51. The method of embodiment 50, wherein the hyperproliferative diseasecomprises one or more of cancer, precancer, or cancer metastasis.52. The method of embodiment 51 or 52, wherein the hyperproliferativedisease comprises one or more of melanoma, lung cancer, breast cancer,pancreatic cancer, brain cancer, prostate cancer, head and neck cancer,kidney cancer, colorectal cancer, lymphoma, colon cancer, or livercancer.53 The method of any one of embodiments 50-52, wherein the administeringcomprises one or more of: injection to or at a tumor in the subject;infusion locally to or at a tumor in the subject; systemic injection inthe subject; systemic infusion in the subject; inhalation by thesubject; oral administration to the subject; or topical application tothe subject.54. The method of any one of embodiments 50-53, wherein theadministering comprises microneedle application.55. A method of enhancing an effect of an anti-cancer therapy in asubject in need thereof, comprising administering to a subject in needthereof an effective amount of the therapeutic construct of any one ofembodiments 1-39 or the composition of embodiment 40, and at least oneanti-cancer agent.56. The method of embodiment 55, wherein the anti-cancer agent is achemotherapeutic agent, a targeted therapeutic agent, or an immunecheckpoint inhibitor.57. The method of embodiment 55 or 56, wherein the therapeutic constructor composition and the anti-cancer agent are administered sequentiallyor concurrently.58. A method of enhancing, increasing, or improving a radiation therapyeffect in a subject diagnosed as having a neoplasia, comprisingadministering to a subject in need thereof an effective amount of thetherapeutic construct of any one of embodiments 1-39 or the compositionof claim 40, and at least one radiation therapy.59. The method of embodiment 58, wherein the therapeutic construct orcomposition and the radiation therapy are administered sequentially orconcurrently.60. The method of any one of embodiments 49-59, wherein the subject ishuman.61. A kit including the immunotherapeutic construct of any one ofembodiments 1-39 and at least one anti-cancer agent.62. The kit of embodiment 61, wherein the anti-cancer agent is achemotherapeutic agent, a targeted therapeutic agent, or an immunecheckpoint inhibitor.

(XI) EXAMPLES Example 1: Combination of PLK1 Inhibition and PD-L1Blockade for Treatment of Cancer

Polo-like kinase 1 (PLK1) is a critical mitotic kinase that isoverexpressed in various cancers and provokes oncogenic properties (Liuet al., Translational Oncology, 10(1): 22-32, 2016). Previous studieshave illustrated the potential of PLK1 inhibition as a therapeuticstrategy and several PLK1 small molecule inhibitors have reachedclinical trials (Gutteridge et al., Molecular Cancer Therapeutics,15(7): 1427-35, 2016). However, PLK1 inhibitors as a monotherapy havenot advanced beyond clinical trials due to poor efficacy anddose-limiting toxicities (de Braud et al. Annals of Oncology: OfficialJournal of ESMO, 26(11): 2341-6, 2015; Schoffski et al., European JCancer, 48(2): 179-86, 2012; Lin et al., British J Cancer, 110(10):2434-40, 2014; Frost et al., Current Oncology, 19(1): e28-35, 2012). Themost advanced PLK1 inhibitor, volasertib (B16727), reached phase IIIclinical trial for acute myeloid leukemia (blood cancer) (Gjertsen etal., Leukemia, 29(1): 11-9, 2015), but eventually failed to meet primaryendpoint of objective response (Ingelheim, Results of Phase III study ofvolasertib for the treatment of acute myeloid leukemia presented atEuropean Hematology Association Annual Meeting. Ridgefield, Conn.,2016). For lung cancer, volasertib was terminated as a monotherapy earlyin a phase clinical trial due to lack of response at the given doselimiting toxicity (300 mg once every 3 weeks) (Ellis et al. , Clinicallung cancer, 16(6): 457-65, 2015). These results suggest thatalternative therapeutic strategies are needed to elicit the fullpotential of inhibiting PLK1.

The recent emergence of immune checkpoint blockade targeting thePD-L1/PD-1 axis have provided promising results for NSCLC patients.PD-L1 expression on tumor cells inhibits tumor directed cytotoxic CD8+ Tcell activity by binding to PD-1 receptor of the T cells and suppressingtheir function (Ohaegbulam et al., Trends in Molecular Medicine, 21(1):24-33, 2015; Shrimali et al., Immunotherapy, 7(7): 777-92, 2015; Zou etal., Science Translational Medicine, 8(328): 328rv4-rv4, 2016).Recently, checkpoint inhibitors for PD-1 and PD-L1 (e.g., pembrolizumab,nivolumab, atezolizumab, and durvalumab) received FDA approval fortreatment of NSCLC, either as first line (pembrolizumab) or second linetherapy (Gettinger, Immunotherapy of advanced non-small cell lung cancerwith immune checkpoint inhibition. (Uptodate.com, 2018). However, whilepatients who respond may show robust and durable responses, only aminority of total patients respond, and many initial responderseventually relapse (Reck et al., New England Journal of Medicine,375(19): 1823-33, 2016; Malhotra et al., Translational Lung CancerResearch, 6(2): 196-211, 2017; Moya-Horno et al., Therapeutic Advancesin Medical Oncology, 10: 1758834017745012, 2018). Furthermore, systemicdistribution of antibodies against immune checkpoints can cause aberrantand uncontrolled immune responses, leading to immune-related adverseeffects (irAEs) that damage normal tissues (Reynolds et al., Journal ofClinical Oncology, 36(15_suppl): 3096, 2018). These toxicities canresult in discontinuation of treatment and in some instances irAEs canbe fatal. Thus, strategies to improve the response and therapeuticefficacy of immune checkpoint blockade are of great interest (Kanwal etal., Cureus, 10(9): e3254-e, 2018).

A recent study showed that PD-L1 protein abundance fluctuated duringcell cycle progression in multiple human cancer cell lines, peaking in Mand early G1 phase (Zhang et al., Nature, 553(7686): 91-5, 2018).Accordingly, increased PD-L1 protein abundance was observed in multiplemouse tumor-derived cell lines arrested in M phase by nocodazole ortaxol (Zhang et al., Nature, 553(7686): 91-5, 2018). Reduction of PLK1induced a strong mitotic arrest that can be sustained for several dayspost treatment (FIGS. 2A-2C, and Morry et al., Mol Cancer Ther. 2017,16(4):763-772). Collectively, these observations led us to hypothesizethat combining PD-L1 antibodies with mitotic kinase inhibitors, such asPLK1 inhibitors, can increase cancer cell killing owing to the apoptoticeffect of the PLK1 inhibitors and the anti-tumor immune effect thatwould be provoked by PD-L1 checkpoint blockade.

Herein, there is described development a PLK1 inhibitor loadedmesoporous silica nanoparticle platform (MSNP) conjugated to PD-L1antibody to synergize combination effects of targeting both PLK1 andPD-L1. By utilizing the nanoparticle construct or antibody drugconjugate (ADC) to co-deliver these agents, we can effectivelyco-localize therapeutic effects to the tumor and reduce toxic concernsassociated with systemic treatment of the drugs. The construct alsotriggers adaptive immunity against cancer. Our study highlights arationale combination strategy to augment existing therapies withoutincreasing toxicity by utilizing MSNP platform as a delivery carrier.

Materials and Methods

Cell lines and reagents: A549 NSCLC were purchased from ATCC (CCL-185)and maintained in RPMI media with 10% fetal bovine serum (FBS). LewisLung Carcinoma (LLC) metastatic variant, LLC-JSP cells, and fluorescentlabeled LLC-JSP cells were gift from Dr. Don Gibbons lab (MD AndersonCancer Center), and were cultured in RPMI+10% FBS. Antibodies used:Human PD-L1 antibody (eBioscience), mouse PD-L1 (PE, BD Biosciences),mouse CD3 (APC, eBioscience), mouse CD8a (Pacific Blue, Invitrogen),mouse CD4 (BV711, BD biosciences), mouse PD-1 (PE/Cy7, BioLegend). AlexaFluor 488 secondary antibody was purchased from Life Technologies. Invivo grade mouse PD-L1 antibody was purchased from BioXcell (BE0101),and volasertib was purchased from Selleckchem. SiRNA sequences: PLK1(antisense 5′-UAUUCAUUCUUCUUGAUCCGG-3′; SEQ ID NO: 2); scrambled SCR(antisense 5′-UUAGUCGACAUGUAAACCA-3′ SEQ ID NO: 3) were purchased fromDharmacon.

Nanoparticle synthesis and characterization: Bare MSNPs were synthesizedas we have previously reported (Ngamcherdtrakul et al., AdvancedFunctional Materials, 25(18): 2646-59, 2015, and U.S. Patent PublicationNo. 2017/0173169). For PLK1 inhibitor loading, volasertib was dissolvedin DMSO and diluted in ethanol solution and mixed with MSNPs in ethanolfor overnight shaking at room temperature (350 RPM). The next day,nanoparticles were coated with PEI (Alfa Aesar) and mal-PEG-NHS (Jenkem)following our previous studies (Ngamcherdtrakul et al., AdvancedFunctional Materials, 25(18): 2646-59, 2015; Ngamcherdtrakul et al., IntJ Nanomedicine, 13: 4015-27, 2018). For PD-L1 antibody conjugation, invivo grade mouse PD-L1 antibody (BioXcell) was buffer exchanged to PBSpH 8 (Zeba spin column, Thermo Fisher) and thiolated using Traut'sreagent (Thermo Fisher) following manufacturer's protocol. Thiolatedantibody was added to NP at 20 wt. % and shaken overnight at 4° C. (300RPM). Nanoparticles were washed with PBS pH 7.2 before characterization.Nanoparticle size was 90 nm, determined using Malvern Zetasizer.Antibody loading was 4 wt. %, determined by protein quantification of NPsupernatant with BCA assay. To quantify PLK1 inhibitor loading,nanoparticles were shaken in DMSO solution to release the drug andsupernatant was collected. Absorbance of supernatant was measured withTecan plate reader to determine loading extent to be 0.5 wt. %. Thep-iPLK1-NP is nanoparticle loaded with both PLK1 inhibitor and PD-L1antibody, p-NP is nanoparticle loaded with PD-L1 antibody, and iPLK1-NPis nanoparticle loaded with PLK1 inhibitor.

Flow cytometry: Cells (100K cells/well) were plated in 6 well platesovernight and treated with indicated treatments the next day. Followingtreatments, cells were collected and aliquoted to 1 million cells persample before washing in FACs buffer and staining. Primary and secondaryantibodies were stained for 30 mins and 1 hour, respectively, underrocking on ice. After staining, cells were washed in FACs buffer beforeflow analysis with Guava easyCyte (Millipore Sigma) flow cytometer(10,000 events per sample). For tumors, tumors were harvested, minced,and incubated with 1 mg/ml DNAse for 30 minutes before smashing through70 μm filter to obtain single cell suspension. RBC lysis buffer wasincubated with cells for 5 minutes, and washed with PBS. 1 million cellsper sample were blocked with Fc-shield before staining with dyeconjugated antibodies for 30 minutes (in FACs buffer). Cells were thenwashed with FACs buffer and analyzed with Guava (50,000 events persample).

Western Blot. Cells were seeded in 6 well plates overnight and treatedwith indicated treatments. Cell culture medium was changed one dayaftertreatment. Three days post treatment, cells were lysed in RIPAbuffer (50-100 μl per well). Lysate was sonicated and centrifuged(15,000 RPM for 15 minutes) and supernatant was collected. Amount oftotal protein was quantified using BCA. 30 μg of proteins (per sample)were mixed with 4× Novex NuPAGE LDS sample buffer andbeta-mercaptoethanol (10% final concentration). Samples were denaturedfor 5 min at 95° C. and loaded onto gel (NuPAGE) for electrophoresis.Proteins were then transferred onto PVDF-FL membrane and blocked withLICOR blocking buffer. Membranes were incubated with primary antibodiesovernight (PLK1, phospho-STAT3 (Tyr705), β-ACTIN) at 4° C. Next day,membranes were rinsed with TBS-T and IRDye conjugated secondaryantibodies (LI-COR) were added for 1 hour under rocking at roomtemperature. Membranes were scanned on a LI-COR Odyssey CLx imagingsystem.

Cell viability after treatments: Cells (1500/well) were plated in whiteflat bottom 96 well plate overnight. The following day, cells weretreated with indicated treatments and media was changed 24 hr posttreatment. 3-5 day post treatment, cell viability was assessed usingCell Titer Glo assay (Promega) following manufacturer's instructions.Luminescence was read with Tecan plate reader.

RT-qPCR to assess PLK1 gene knock down: RNA was isolated with GeneJetRNA purification kit (Thermo Fisher Scientific) following manufacturer'sinstructions. One-Step qRT-PCR was performed using EXPRESS One-StepSuperscript™ qRT-PCR Kit (Invitrogen). Cycling conditions: 50° C. for 2min, 95° C. for 10 min, 40 cycles of 95° C. for 15 s, and 60° C. for 1min. TAQMAN gene expression primers Human HPRT mRNA (Hs99999909_m1),Human PLK1 mRNA (Hs00983225_g1), and Human PD-L1 (Hs00204257_ml) wereused. Data was analyzed using 2^(−ΔΔC(t)) method.

Syngenic tumor models and treatments: For single tumor flank model,LLC-JSP murine lung cancer cells (200K) were inoculated in right flankof C57BL/6 female mice (6 weeks) (Charles River NCI colony). At 8 dayspost tumor inoculation, mice received intraperitoneal (i.p.) treatmentsof volasertib (20 mg/kg) and/or PD-L1 antibody (mouse PD-L1 fromBioXcell; 10 mg/kg) every 5 days for 3 doses total. Tumors were measuredwith Vernier Caliper and volume calculated by V=0.5×length×width². Forbilateral tumors, C57BL/6 were inoculated with 100K and 40K LLC-JSPcells in right and left flank, respectively. At 12 days postinoculation, the aforementioned treatments were administeredintratumorally to the right tumor every 3 days for 3 doses total. Forboth single flank and bilateral flank tumor models, mice were sacrificedwhen total tumor burden exceeded 2000 mm³. For metastatic lung tumormodel, LLC-JSP (200K) were injected intravenously (i.v.) to 6 week oldC57BL/6 mice. At 3 days post cancer cell injection, mice were randomlygrouped and treated with i.v. saline, p-iPLK1-NP (25 mg/kg NP), or i.p.PD-L1 antibody (5 mg/kg) and volasertib (1.25 mg/kg) every 3 days for atotal of 4 doses. All studies were reviewed and approved byInstitutional Animal Care and Use Committee (IACUC) at Oregon Health andScience University (OHSU).

Statistical analysis: GraphPad Prism 8.0 (GraphPad Software Inc.) wasused for all statistical analysis. Comparison between two groups wasperformed with Student's t test. Tumor growth was analyzed using two-wayrepeated measures ANOVA with Tukey's correction for multiplecomparisons. Kaplan Meier survival curve was analyzed using the log-rank(Mantel-Cox) method. Significance was set at p<0.05. In vitro data areexpressed as mean±SD; in vivo data are expressed as mean±SEM.

Results

PLK1 knock-down induces expression of PD-L1 in cancer cells: Mitotickinase inhibitors such as small molecule inhibitor (e.g., BI2536) orsiRNA against PLK1 delivered on a nanoparticle (see Patent PublicationNo. 2017/0173169) which knocked down PLK1 (FIGS. 2A-2B), leading to lungcancer cell death (FIG. 2C), and putting the cancer cells in G2/M growtharrest (FIG. 2D). This agrees with a previous report that PLK1inhibition or knock-down results in cell cycle arrest in G2/M in breastcancer (Morry et al., Mol Cancer Ther., 16(4):763-772, 2017).

The PLK1 knockdown resulted in an increase in PD-L1 surface expressionin both human (A549, FIGS. 3A-3B) and murine (LLC-JSP, FIG. 3C) lungcancer cell lines. As shown in FIG. 3A, 85% knockdown of PLK1 mRNA (bysiRNA against PLK1) resulted in 2.5-fold increase in PD-L1 mRNAexpression in A549 cell line compared with control treated cells. Thiswas then confirmed at the surface protein level in A549 (FIG. 3B) andLLC-JSP (FIG. 3C) lung cancer cell lines at 3 days post siRNAtreatments.

Mitotic kinase inhibitors kill cancer cells and upregulate PD-L1expression. Following on our discovery that PLK1 knockdown results inPD-L1 upregulation, we sought to determine whether this holds true forinhibition of mitotic kinases in general. Three leading mitotic kinaseinhibitors screened against PLK1 (volasertib), Aurora kinase A(alisertib), and CHK1 (AZD7762) in mouse lung cancer cell lines. Asshown in FIG. 4, treatment of LLC-JSP (a murine lung cancer cell line)with volasertib, alisertib, or AZD7762 led to significant cell death(FIG. 4A) and upregulated surface PD-L1 level (FIGS. 4B-4C) in eachcase. This confirmed the link between mitotic kinase inhibition(regardless of the kinase classes) and PD-L1 upregulation. The survivingcells have increased levels of immune checkpoint molecules (e.g., PD-L1,FIGS. 3A-3C and FIGS. 4B-4C), which prevents cytotoxic T cells fromattacking the surviving cancer cells. Thus, co-delivery of a mitoticinhibitor (e.g., PLKs, Aurora kinases, CHK1, CDK1/2, HASPIN, Mps1, NEKinhibitors) and immune checkpoint inhibitor (e.g., monoclonal antibodyagainst PD-L1, PD-1, CTLA-4) on the same construct will yield greatercancer death.

Combination of PLK1 inhibition with PD-L1 blockade enhances tumorcontrol in vivo: Based on our finding that PLK1 reduction results inPD-L1 increase, we sought to investigate whether PLK1 inhibition andPD-L1 blockade would synergize in vivo. We used LLC-JSP cell line todevelop flank tumor model in immune-competent mice (Chen et al., NatureCommunications, 5: 5241, 2014). Established tumors (>60 mm³) at day 8post tumor inoculation were treated i.p. with the PLK1 inhibitorvolasertib (20 mg/kg) and PD-L1 monoclonal antibody (10 mg/kg) every 5days for a total of 3 doses (FIG. 5A). As shown in FIG. 5B, thecombination treatment significantly reduced tumor growth compared withsingle drug administrations. Moreover, the combination significantlyprolonged survival of mice (FIG. 5C), confirming our hypothesis.

Nanoparticle delivery of PLK1 inhibitor volasertib (iPLK1-NP): Despitethe promise of mitotic kinase PLK1 as a therapeutic target, clinicaltrials with current small molecule inhibitors have been disappointing.All six of PLK1 inhibitors (GSK461364, BI2536, Tak960, NMS-P937,TKM-PLK1, and B16727) have failed in clinical trials. To reduce toxicityand improve tumor bioavailability of PLK1 inhibitor, we investigatedwhether our MSNP platform could improve the efficacy of a clinicallyavailable PLK1 inhibitor. Morry et al. (Mol Cancer Ther., 16(4):763-772,2017) demonstrated the promise of this MSNP platform to target anddeliver siRNA to breast tumors including those metastasized to lungs andorthotopic lung tumors. In this research, we utilized the platform todeliver the small molecule inhibitor volasertib, which is the mostadvanced inhibitor of PLK1. Volasertib was loaded onto mesoporous silica(FIG. 6A) prior to surface modification with polyethylene imine (PEI)and polyethylene glycol (PEG). The final nanoparticle (referred to asiPLK1-NP) size is 90 nm (FIG. 6B) which is in the appropriate range totake advantage of the EPR effect, and contains 0.5 wt. % PLK1 inhibitorvolasertib. As shown in FIG. 6C, treatment of LLC-JSP cells withvolasertib or iPLK1-NP significantly reduced cell viability comparedwith vehicle treated cells in a dose-dependent manner. Further,treatment with iPLK1-NP reduced cell viability more than the free PLK1inhibitor (FIG. 6C). In agreement with previous finding using PLK1 siRNA(FIGS. 3A-3C) and mitotic kinase inhibitors (FIGS. 4B-4C), treatmentwith iPLK1-NP resulted in significant increase in PD-L1 cell surfaceexpression (FIG. 6D).

Nanoparticle for co-delivery of iPLK1 and PD-L1 antibody (p-iPLK1-NP):As iPLK1-NP could effectively kill cancer cells and simultaneouslyupregulate PD-L1 of the surviving cells, we aimed to utilize this as anadvantage to target PD-L1+ cancer cells by conjugating PD-L1 antibody oniPLK1-NP. In this sense, a feed-forward loop can be generated whererepeated administrations of PD-L1 targeted iPLK1-NP (referred to asp-iPLK1-NP) would upregulate PD-L1 expression and allow for superiortumor targeting to induce both apoptosis (via PLK1 inhibition) andanti-tumor immune responses (via PD-L1 blockade). This would beparticularly advantageous for treating tumors without obvioustargets/receptors for nanoparticle delivery, and may ultimately allowfor higher response rates of immune checkpoint blockade. As illustratedin FIG. 7A, PD-L1 antibody was conjugated to PEG on NPs, and antibodyamount was determined by BCA assay to be 4 wt. %. The hydrodynamic sizeof the construct is shown in FIG. 7B to be about 90 nm. As withiPLK1-NP, treatment with p-iPLK1-NP significantly reduced cell viabilityin LLC-JSP cell line (FIG. 7C). Furthermore, LLC-JSP cells incubated for2 hours with p-iPLK1-NP blocked PD-L1 surface receptors as much as freePD-L1 antibody delivered at 25-fold higher dose (FIG. 7D). This islikely due to the high local concentration of antibody the cellexperienced when antibody was delivered with nanoparticles. The iPLK1-NPhad no effect on PD-L1 level at this short time point (FIG. 7D).Treatment of cells for 2 days with iPLK1-NP increased PD-L1 level asanticipated, which was reduced to normal level (see untreat) upontreatment with nanoparticle containing PD-L1 antibody (p-iPLK1-NP) (FIG.7E). This demonstrates the nanoparticle targeting and blockade of PD-L1receptors, which are induced by PLK1 inhibition.

Local delivery of p-iPLK1-NP reduces local and distant tumor growth: Toassess the anti-tumor immune response of p-iPLK1-NP, we utilized abilateral flank tumor model. C57BL/6 mice were injected with 100K and40K LLC-JSP cells on the right and left flank, respectively. At day 12post injection, the right flank (local) tumors were injected with PBS,p-NP, iPLK1-NP, or p-iPLK1-NP (0.5 mg NP, 2.5 μg iPLK1, 20 μg PD-L1)every 3 days for a total of 3 injections (FIG. 8A). Tumor growth oflocal (treated) and distant (untreated) tumors were monitored.Treatments with p-iPLK1-NP significantly reduced tumor growth of localtumor compared with nanoparticle containing a single drug (p-NP oriPLK1-NP) (FIG. 8B). Importantly, a delay in the onset of distant tumorswas also observed for p-iPLK1-NP treated mice (FIG. 8C), whichillustrates that an anti-tumor immune response was generated. Theantitumor immune effects did not come from PD-L1 on nanoparticle alonebut were contributed by both PD-L1 and PLK1 inhibitors on thenanoparticle (FIG. 8C). Furthermore, treatment of p-iPLK1-NPsignificantly prolonged survival of mice compared with saline control orsingle drug NPs (FIG. 8D). Additionally, in a separate study, mice wereinjected with saline or p-iPLK1-NP as illustrated in FIG. 8A and tumorswere harvested one day after last treatment to assess T cellinfiltration. As shown in FIG. 8E, tumors treated with p-iPLK1-NP hadsignificantly higher CD3+ and CD8+ tumor infiltrating lymphocytes(TILs), while CD4+ TILs were not enhanced compared with the controltumors.

Systemic administration of p-iPLK1-NP prolongs survival of mice withexperimental metastatic tumors: To demonstrate the clinical potential ofp-iPLK1-NP for lung cancer, we developed an experimental metastatic lungtumor model by intravenous injection of LLC-JSP cells (200K cells).Three days post cell injection, mice were randomly grouped and treatedwith saline, p-iPLK1-NP, or free drugs (volasertib+PD-L1 antibody) every3 days for 4 doses total, as shown in FIG. 9A. The free drugs wereadministered at 5-fold higher dose than the amounts on NP. Mice treatedwith p-iPLK1-NP survived significantly longer than those treated withsaline (FIG. 9B). The presence of lung tumor was confirmed visually foreach deceased mouse. Data indicate that p-iPLK1-NP was as effective asthe free drugs administered at 5-fold higher dose owing to the abilityof nanoparticles for tumor targeting and co-localizing the therapeuticeffects as well as triggering adaptive antitumor immunity. Furthermore,treatment with p-iPLK1-NP did not cause any weight loss, demonstratingthe safety of the construct (FIG. 9C). Systemic applications of thetherapeutic construct allow the treatment of other cancers that are notapplicable for local delivery.

Systemic administration of p-iPLK1-NP is dependent on CD8+ T cells:C57BL/6 mice were injected with 200K LLC-JSP cells intravenously. After3 days, mice were treated with saline, p-iPLK1-NP (i.v., containing 2.5μg volasertib and 20 μg PD-L1 antibody), or p-iPLK1-NP+anti-CD8 (200 μgtwice weekly). As shown in FIG. 10, the efficacy of p-iPLK1-NP wasimmune-mediated as CD8 depletion abolished the effects of p-iPLK1-NP andprolonged survival was not observed (saline vs. p-iPLK1-NP+anti-CD8antibody; p>0.05=ns). Immune mediated response supersedes direct drugeffect with this specific nanoconstruct.

Feed-forward (positive feedback loop) delivery and specificity ofanti-PD-L1 conjugated NP. While p-iPLK1-NP initially reduces PD-L1levels upon binding and internalization (as shown in FIG. 7D-E),surviving cells have upregulated PD-L1 level (FIG. 6D) due to thesignaling effects of PLK1 inhibition. In this context, unregulated PD-L1is used as the homing target for subsequent delivery of p-iPLK1-NP,leading to cancer targeting in a feedforward manner (i.e., highertargeting as increased doses of the treatment until all cancer is gone).To investigate the feedforward targeting of p-iPLK1-NP, we used 4T1murine cancer cells which express low baseline PD-L1 levels. p-iPLK1-NPled to the upregulation of PD-L1 in 4T1 cells 4 days post treatment(FIG. 11A). We then assessed the cellular uptake of p-iPLK1-NP incontrol (with low PD-L1) and p-iPLK1-NP-treated 4T1 cells (withupregulated PD-L1). As shown in FIG. 11B, after 1 hour of exposure,p-iPLK1-NP was preferentially taken up by the PD-L1 high cells vs. PD-L1low cells by nearly 4-fold, demonstrating the selectivity andfeed-forward targeting by p-iPLK1-NP. We also evaluated the cell killingselectivity by comparing viability of various cancer cells (lungLLC-JSP, breast 4T1, melanoma B16-F10 cancer cells) vs. bonemarrow-derived dendritic cells (BMDC) after treatment with p-iPLK1-NP.As shown in FIG. 11C, p-iPLK1-NP led to significant cell killing incancer cells but minimal killing in dendritic cells, needed for antigenpresentation to develop anti-tumor T cells. Inhibition of mitotickinases such as PLK1 (e.g., with siRNA, FIG. 12A) also reducedphosphorylation of STAT3, thus would be beneficial to modulatingimmunosuppressive environment of tumors.

DISCUSSION

In this example, it is shown that inhibition of PLK1 and other mitotickinases Aurora Kinase A and CHK1 results in an increase of immunecheckpoint PD-L1 expression in human and mouse NSCLC cells. Thissuggests that avoiding the immune response is a mechanism exploited bycancer cells that survive mitotic kinase inhibition.

Previous studies have also shown roles of PLK1 in regards to immunity.For instance, PLK1 has been shown to be a regulator of STAT3 activation(Zhang et al., Gastroenterology, 142(3): 521-30.e3, 2012), whichpromotes an immune suppressive microenvironment, and inhibiting PLK1resulted in loss of phosphorylated STAT3 in NSCLC cells (Yan et al.,Oncology Letters, 16(5): 6801-7, 2018) and as reported herein (FIG.12A). Further, PLK1 was found to associate with the MAVS and negativelycontrols its activity in inducing type I interferons (Gringhuis et al.,Nature Immunology, 18(2): 225-35, 2017; Vitour et al., J BiologicalChemistry, 284(33): 21797-809, 2009). Intriguingly, PLK1 inhibition hasalso been shown to significantly increase HLA mRNA which encode MHCclass I protein, the antigen presenting surface receptors (Li et al.,Journal of Oncology, 2018: 3979527, 2018). These studies suggest thatPLK1 inhibition may be promising to augment immunotherapy. However, tothe best of our knowledge, this is the first study to report theeffectiveness of the combination of PLK1 inhibition with immunotherapy.

PLK1 inhibition induces PD-L1 upregulation and co-delivery of PD-L1antibodies and PLK1 inhibitors significantly enhance the treatmentoutcome as shown in for NSCLC. Other cytotoxic agents have also beenshown to increase PD-L1 expression, including paclitaxel in ovariancancer (Peng et al., Cancer Research, 75(23): 5034-45, 2015), CDK4/6inhibitors (Zhang et al., Nature, 553(7686): 91-5, 2018), and PARPinhibitors (Jiao et al., Clin Cancer Res, 23(14): 3711-20, 2017) inbreast cancer. Therefore, it is logical that these drugs are now inclinical investigations in combination with PD-L1 checkpoint blockade(Esteva et al., The Lancet Oncology, 20(3): e175-e86, 2019). Ourfindings also suggest that these and other cytotoxic agents can becombined with PD-L1 immune checkpoint blockade on our nanoparticles tofacilitate effective therapy and reduce toxicity in clinics.

The research presented in this example focused on lung cancer, theleading cancer killer (American Cancer Society, Cancer Facts & Figures.2018). Like melanoma, where immunotherapy has been the most promising,lung cancer is a disease with a high mutational load which drives theexpression of various neo-epitopes which can be recognized by hostimmune system (Campbell et al., Nature Genetics, 48(6): 607-16, 2016;Rizvi et al., Science, 348(6230): 124-8, 2015). Consequently,immunotherapy is a promising approach to treat lung cancer. However,objective response rates are much lower for lung cancer patients thanmelanoma. The research described here illustrates how superior responsescan be achieved for lung cancers when combining PLK1 inhibition withPD-L1 blockade. Further, as the increase of PD-L1 is not specific toPLK1 inhibitors, other cytotoxic agents that induce upregulation ofPD-L1 can be explored to synergize with current immune checkpointblockade agents. Additionally, by co-localizing therapeutic effects withour MSNP platform, the dose of the drugs required can be reduced by5-fold. This suggests that nanoparticles can improve efficacy and reducesystemic toxicities of free drugs. This is key to improving outcomes ascurrent combination therapy strategies with immune checkpoint blockadecan lead to higher rates of adverse events. Lastly, due to theversatility of the MSNP platform, siRNA can also be loaded to target anygene identified as a regulator of cancer progression or immune evasion,in addition to the targeting antibody (e.g. PD-L1) and PLK1 inhibitor.

Example 2: Adjuvant Oligonucleotides to Enhance Therapeutic ConstructFunction

Adjuvant oligonucleotides can also be incorporated to enhance anti-tumorimmunity. For instance, incorporation of CpG on p-iPLK1-NP (referred toas p-iPLK1-NP-CpG) significantly improved survival of 2 out of 7 mice,and one mouse was completely free of tumors (FIG. 13). CpGoligodeoxynucleotides act as a danger associated molecular pattern(DAMP) to stimulate PRR, specifically the toll-like receptor 9 (TLR9).This serves as a danger signal for the activation of antigen presentingcells and subsequent priming of T cells. Thus, by releasing antigens(via cancer killing by mitotic inhibitor), delivering CpG adjuvant, andblocking immune checkpoints, this therapeutic tackles various strategiesby which cancer cells evade the immune response (Patel & Minn, Immunity48(3):417-433, 2018).

Example 3: Antibody-Drug Conjugate (ADC) of Alisertib and PD-L1 AntibodyLead to Enhance Cell Killing Compared to Free Drug Counterparts

Immune checkpoint antibody-mitotic kinase inhibitor ADC is composed ofan immune checkpoint antibody, an MKI, and linkers. The antibody servesas the carrier for drugs (MKIs). The linkers can be tailored to getdesired ADC's physicochemical properties and pharmacokinetics and tocontrol the drug liberation. The drugs can be release outside or insidetargeted cells. If the drug is released inside the targeted cells, theantibody also serves as the targeting moiety to enhance the drug uptakeinto cells. The drug-to-antibody ratio may range from 1 to 20. Idealratio (e.g., about 2 to 8 or about 4 to 6) should yield bestpharmacokinetics and tumor accumulation, as well as highest antitumoractivities.

MATERIALS AND METHODS. The synthesis of PD-L1-alisertib contained 3steps. (1) Alisertib-PEG conjugation, (2) PD-L1 activation, and (3)PD-L1-alisertib conjugation. (1): 0.3 ml of 5 mg/ml alisertib (SelleckChemicals, USA) in dimethyl sulfoxide (DMSO) (Fisher Scientific, USA)was mixed with 50 μl of 60 mg/ml(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (ThermoScientific, USA) in DMSO and 27 μl of 60 mg/ml N-hydroxysuccinimide(Sigma Aldrich, USA) in DMSO. After that, 57.8 μl of 40 mg/ml H2N-PEG-SH(MW 400) (Nanocs, USA) in DMSO was added. The reactant mixture waspurged with N2 for 1 minute and then stirred at room temperature for 12hours. Distilled water was then added to precipitate alisertib-PEG-SH.Alisertib-PEG-SH was collected by centrifuge at 15,000 rpm, 4° C. for 10minutes and washed 3 times with distilled water. The final cleanalisertib-PEG-SH was lyophilized (AdVantage 2.0, SP Scientific, USA) forlong term storage. (2): 0.147 ml of 6.76 mg/ml PD-L1 (BioXCell, USA) wasmixed with 0.9 ml of phosphate buffer saline (PBS) (pH 7.2) and 29 μl of5 mg/ml sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC) (Thermo Fisher, USA) solution in water. The reactantmixture was stirred at room temperature for 1 hour. The pure PD-L1-SMCCwas collected by using a desalting column (Thermo Fisher, USA). (3): 750μg of PD-L1-SMCC and 70 μg of alisertib-PEG-SH were dissolved in amixture of PBS (pH 7.2) (1 ml), DMSO (50 μl), and propylene glycol (50μl). The reactant mixture was purged with N2 for 1 minute and thenstirred at room temperature for 24 hours. The PD-L1-alisertib solutionwas collected by a desalting column and then lyophilized. Thedrug-to-antibody ratio was determined by UV absorbance at 280 nm and 318nm.

RESULTS: We have prepared PD-L1 antibody-alisertib conjugates usingN-(maleimidomethyl) cyclohexane-1-carboxylate (MCC) linker used in anFDA-approved ADC drug (Kadcyla or T-DM1). MCC linker is not cleavable,but, before conjugation to antibody-MCC, alisertib was modified with ashort PEG chain via —HN—CO— bond (FIG. 14A). This amide bond could behydrolyzed by acidic conditions in endosomes and lysosomes, releasingalisertib intact. The short PEG₈ (MW 400 Da) was used to enhance thesolubility of alisertib and introduce a thiol group for conjugation withantibody-MCC. Desalting columns were used for removing free alisertiband thiolated alisertib. The drug-to-antibody ratio (DAR) was 6alisertib molecules per antibody (determined by UV-Vis). The ADC hadsignificantly greater cytotoxicity in LLC-JSP cells when compared tofree alisertib (FIG. 14B), while free PD-L1 did not have any effects onthe cell viability (FIG. 14C).

Example 4: Topical Formulation and Application of Therapeutic Construct

The therapeutic constructs disclosed herein can be formulated intotopical formulations. Several vehicles known in the art can be mixedwith the construct, e.g., Aquaphor (ointment-based) and Carbopol(gel-based). Heat or surfactant (e.g., Polysorbate 80 (Tween 80) as anemulsifier) can be used to allow better mixing of the vehicle and anaqueous suspension of AIRISE. As an example, it was confirmed that 10wt. % Tween-80 did not cause any premature leakage of siRNA from thenanoparticle. It was also shown that 2.5 wt. % Tween-80 was sufficientto enhance the mixing of siRNA-NP and Aquaphor upon warming the mixtureto 55° C.

Methods to enhance penetration simultaneously can be used, such asultrasound and microneedle rollers (e.g., Dermaroller® with the needleheight ranging from 0.5 mm to 1.5 mm). Application of microneedles withneedle height as short as 0.5 mm can enhance penetration of topicalsiRNA-nanoparticle formulation when tested in pig skin (FIG. 15) and inmice (FIG. 16).

FIG. 15 shows that microneedle roller enhances penetration of siRNAnanoparticle construct when tested in pig skin, which is similar inthickness to human skin. Pig skins were incubated with the formulation(Dy677-siSCR-NP in Aquaphor) for 1.5 h (37° C.; 5% CO₂). After 1.5hours, a skin punch was taken from the treated area and processed forfluorescent imaging using a standard approach. Significant enhancementin skin penetration with a microneedle roller was observed. While siRNAsignal (arrows) was confined to the outer surface of the pig skin whensiRNA-NP in Aquaphor was given without a roller, we observed siRNAsignal (arrows) past the epidermis down to the dermis layer withmicroneedle pre-application (FIG. 15).

FIG. 16 shows that microneedle roller enhances topical delivery ofsiRNA-NP. First, mice were shaved one day before treatment.Dy677-siSCR-NP (0.72 nmol siRNA) was mixed with 100 μL of 2.5%Tween-Aquaphor (per one application). Right before treatment, a dermalmicroroller was applied to only one side of the back in four directionsconsistently, while the other side was not pre-treated. The mixture wasapplied to the shaved area (approximately 2 cm² application area) withand without microneedle pre-treatments for comparison. After 1.5 hr oftreatment time, treated skin samples were harvested and processed forimaging.

FIGS. 17A-17B show the resulting gene knockdown at 3 days aftermicroroller+topical siRNA nanoconstruct application. A 55% EGFRknockdown in siEGFR-NP versus saline treated group (*p<0.05) (FIG. 17A)was observed. In comparison, one intradermal injection of siEGFR-NP(with same siEGFR dose of 0.72 nmol) resulted in 40% EGFR knock downversus saline treated groups (FIG. 17B). Microneedle form of therapeuticconstruct. The use of dissolvable microneedles based on dextran,amylopectin, PVP, PEG, methylcellulose, chitosan, or other polymers orcompounds known in the arts were explored for microneedle fabrications,as shown in FIG. 18, which allow for painless in-home treatment and arehighly effective at delivering AIRISE-02 owing to high needle density(100 needles per 1 cm²). As an example (FIG. 18), a dextran solution(300 mg/ml in water) containing NP loaded with Dy677-conjugated siRNAwas cast onto a microneedle mold. The solution was centrifuged orvacuumed to fill the mold compactly. The microneedle was dried by air,desiccator, vacuum oven, fridge, or combination thereof and removed fromthe mold. Heights of the needles varied from 300 to 800 micronsdepending on the templates and optimization. siRNA-NP was successfullyloaded into these needle arrays (at about 0.5 nmol siRNA per array) andthe needles were fully dissolved within 5 min after applying to pigskins. Different dissolving time can be engineered by varying theingredients of the microneedles. Microneedle patches of different shapeand forms can also be manufactured with different templates.

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of orconsist of its particular stated element, step, ingredient or component.Thus, the terms “include” or “including” should be interpreted torecite: “comprise, consist of, or consist essentially of.” Thetransition term “comprise” or “comprises” means includes, but is notlimited to, and allows for the inclusion of unspecified elements, steps,ingredients, or components, even in major amounts. The transitionalphrase “consisting of” excludes any element, step, ingredient orcomponent not specified. The transition phrase “consisting essentiallyof” limits the scope of the embodiment to the specified elements, steps,ingredients or components and to those that do not materially affect theembodiment. A material effect, in this context, is a measurablereduction in a biological impact (such as an anti-cancer effect) of atherapeutic construct.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range, i.e.denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents, printedpublications, journal articles and other written text throughout thisspecification (referenced materials herein). Each of the referencedmaterials are individually incorporated herein by reference in theirentirety for their referenced teaching.

It is to be understood that the embodiments of the invention disclosedherein are illustrative of the principles of the present invention.Other modifications that may be employed are within the scope of theinvention. Thus, by way of example, but not of limitation, alternativeconfigurations of the present invention may be utilized in accordancewith the teachings herein. Accordingly, the present invention is notlimited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the example(s) or when application of themeaning renders any construction meaningless or essentially meaningless.In cases where the construction of the term would render it meaninglessor essentially meaningless, the definition should be taken fromWebster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

What is claimed is:
 1. A therapeutic construct comprising: a deliverysystem comprising at least one mitotic kinase inhibitor; and at leastone immune checkpoint inhibitor.
 2. The therapeutic construct of claim1, wherein the delivery system comprises a liposome, a lipid-basedparticle, a polymeric particle, an inorganic or organic nanoparticle ormicroparticle, or a hybrid thereof.
 3. The therapeutic construct ofclaim 2, wherein the delivery vehicle comprises one or more offullerenes, endohedral metallofullerenes, trimetallic nitride templatedendohedral metallofullerenes, single-walled and multi-walled carbonnanotubes, calcium phosphate particles, aluminum salt particles,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, microparticles, quantum dots, superparamagneticnanoparticles, nanorods, cellulose nanoparticles, silicon, silica andpolymer micro- and nano-spheres, silica-shells, biodegradable PLGAmicro- and nano-spheres, gold nanoparticles, cerium oxide particles,zinc oxide particles, silver nanoparticles, carbon nanoparticles, ironnanoparticles, and/or modified micelles.
 4. The therapeutic construct ofany one of claims 1-3, wherein the delivery vehicle comprises amesoporous silica nanoparticle.
 5. The therapeutic construct of claim 4,wherein the mesoporous silica nanoparticle has a mean particle size ofabout 5-200 nm.
 6. The therapeutic construct of claim 4 or 5, whereinthe mesoporous silica nanoparticle is coated with cross-linkedpolyethyleneimine and polyethylene glycol.
 7. The therapeutic constructof any one of claims 1-6, wherein the at least one mitotic kinaseinhibitor and/or immune checkpoint inhibitor comprises anoligonucleotide, a polynucleotide, a small molecule inhibitor, or anantibody.
 8. The therapeutic construct of any one of claims 1-7, whereinthe at least one mitotic kinase inhibitor is an inhibitor of a polo-likekinase (PLK), an Aurora kinase, cyclin-dependent kinase (CDK)1, CDK2,HASPIN, monopolar spindle 1 kinase (Mps1), a NimA-related kinase (NEK).9. The therapeutic construct of any one of claims 1-8, wherein the atleast one mitotic kinase inhibitor comprises one or more of GSK461364,BI2536, Tak960, NMS-P937, volasertib, Chk 1 Kinase Inhibitor LY2603618,AU14022, YK-4-279, AZ703, alisertib, prexasertib, or AZD7762.
 10. Thetherapeutic construct of any one of claims 1-9, wherein the at least onemitotic kinase inhibitor comprises volasertib.
 11. The therapeuticconstruct of any one of claims 1-10, wherein the at least one immunecheckpoint inhibitor comprises a siRNA, inhibitor, or antibody againstone or more of PD-L1, PD-1, TIM-3, LAG-3, or CTLA-4.
 12. The therapeuticconstruct of any one of claims 1-11, wherein the at least one immunecheckpoint inhibitor is an antibody against PD-L1, PD-1, or CTLA-4. 13.The therapeutic construct of any one of claims 1-12, wherein the atleast one immune checkpoint inhibitor is an antibody against PD-1. 14.The therapeutic construct of claim 13, wherein the at least one immunecheckpoint inhibitor comprises at least one of: nivolumab,pembrolizumab, MPDL3280A, ipilimumab, tremelimumab, atezolizumab,avelumab, durvalumab, cemiplimab, pidilizumab, or spartalizumab.
 15. Thetherapeutic construct of any of the previous claims, further comprisingan adjuvant.
 16. The therapeutic construct of claim 15, wherein theadjuvant comprises one or more of a CpG oligonucleotide, a DNA TLRagonist containing a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLRagonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, acytokine, a small molecule TLR agonist, an oil- or surfactant-basedadjuvant, a lipopolysaccharide, a plant extract, or a derivativethereof.
 17. The therapeutic construct of claim 15 or 16, wherein theadjuvant comprises a CpG oligonucleotide, imiquimod, resiquimod,gardiquimod, poly I:C, poly ICLC, dSLIM, or EnanDIM.
 18. The therapeuticconstruct of claim 16, wherein the adjuvant comprises a CpGoligonucleotide.
 19. The therapeutic construct of any one of claims1-18, having a hydrodynamic size of 5-999 nm.
 20. The therapeuticconstruct of any one of claims 1-18, having a hydrodynamic size of1-1000 microns.
 21. A therapeutic construct comprising: an immunecheckpoint inhibitor; a mitotic kinase inhibitor; and a chemical linkerlinking the immune checkpoint inhibitor and the mitotic kinaseinhibitor.
 22. The therapeutic construct of claim 21, wherein themitotic kinase inhibitor is an oligonucleotide, a polynucleotide, asmall molecule inhibitor, or an antibody.
 23. The therapeutic constructof claim 21 or 22, wherein the immune checkpoint inhibitor is anoligonucleotide, a polynucleotide, a small molecule inhibitor, or anantibody.
 24. The therapeutic construct of any one of claims 21-23,wherein the immune checkpoint inhibitor is an antibody.
 25. Thetherapeutic construct of any one of claims 21-24, wherein the immunecheckpoint inhibitor is an antibody against PD-L1, PD-1, TIM-3, LAG-3,or CTLA-4.
 26. The therapeutic construct of any one of claims 21-25,wherein the immune checkpoint inhibitor is an antibody against PD-L1,PD-1, or CTLA-4.
 27. The therapeutic construct of any one of claims21-26, wherein the immune checkpoint inhibitor is an antibody againstPD-L1.
 28. The therapeutic construct of any one of claims 21-27, whereinthe mitotic kinase inhibitor is selected from GSK461364, BI2536, Tak960,NMS-P937, volasertib, Chk 1 Kinase Inhibitor LY2603618, AU14022,YK-4-279, AZ703, alisertib, prexasertib, or AZD7762.
 29. The therapeuticconstruct of any one of claims 21-28, wherein the mitotic kinaseinhibitor is alisertib.
 30. The therapeutic construct of any one ofclaims 21-29, wherein the chemical linker comprises one or more of the ahydrazine; a disulfide; N-succinimidyl-4-(2-pyridyldithio)butanoate;N-succinimidyl-4-(2-pyridyldithio)-2-sulfo butanoate; perfluorophenyl3-(pyridin-2-yldisulfanyl)propanoate; 2,5-dioxopyrrolidin-1-yl3-methyl-3-(pyridin-2-yldisulfanyl)butanoate; Gly-Phe-Leu-Gly;Ala-Leu-Ala-Leu; Val-Cit; Phe-Lys; Val-Ala; Ala-Phe-Lys; Phe-Lys;(Gly)_(n), wherein n is 1-20; a β-glucuronide linker; maleimidocaproyl;N-(maleimidomethyl)cyclohexane-1-carboxylate;4-(4-acetylphenoxy)butanoic acid; dibromomaleimide; para-aminobenzoicacid; 4-nitrophenol; acetic acid; formic acid; 4-maleimidobutyric acidN-succinimidyl ester; N-(4-maleimidobutyryloxy)succinimide;N-(6-maleimidocaproyloxy)succinimide; 3-maleimidopropionic acidN-succinimidyl ester; N-(3-maleimidopropionyloxy)succinimide;5-maleimidovalericacid-NHS; linear, branched, or multi-arm polyethyleneglycol having a molecular weight of 100-10000 Da;propargyl-N-hydroxysuccinimidyl ester; pyrophosphate;succimimidyl-4-azidobutyrate; 4-azidobenzoic acid N-hydroxysuccinimideester; tert-butyl1-(4-formylphenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-oate; or aresidue thereof.
 31. The therapeutic construct of any one of claims21-30, wherein the chemical linker comprisesN-(maleimidomethyl)cyclohexane-1-carboxylate linker or a residuethereof.
 32. The therapeutic construct of any one of claims 21-31,wherein the chemical linker comprises sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
 33. The therapeuticconstruct of any one of claims 21-32, wherein the chemical linkercomprises a linear polyethyleneglycol having a molecular weight of100-10000 Da, or a residue thereof.
 34. The therapeutic construct of anyone of claims 21-33, wherein the ratio of mitotic kinase inhibitor toimmune checkpoint inhibitor is about 1-20.
 35. The therapeutic constructof claim 34, wherein the ratio of mitotic kinase inhibitor to immunecheckpoint inhibitor is about 2-8.
 36. The therapeutic construct ofclaim 34 or 35, wherein the ratio of mitotic kinase inhibitor to immunecheckpoint inhibitor is about 4-6.
 37. The therapeutic construct of anyone of claims 21-33, wherein the ratio of immune checkpoint inhibitor tomitotic kinase inhibitor is about 1-20.
 38. The therapeutic construct ofclaim 37, wherein the ratio of immune checkpoint inhibitor to mitotickinase inhibitor is about 2-8.
 39. The therapeutic construct of claim 37or 38, wherein the ratio of immune checkpoint inhibitor to mitotickinase inhibitor is about 4-6.
 40. A composition comprising thetherapeutic construct of any one of claims 1-39 and a pharmaceuticallyacceptable carrier, excipient, or diluent.
 41. A method of treatingcancer comprising administering to a subject with cancer an effectiveamount of the therapeutic construct of any one of claims 1-39, or thecomposition of claim
 40. 42. The method of claim 41, wherein the subjectis a human.
 43. A method of treating a cell exhibiting symptoms ofcancer comprising contacting the cell with a therapeutically effectiveamount of the therapeutic construct of any one of claims 1-39, or acomposition of claim
 40. 44. A method of treating a cell obtained from asubject exhibiting symptoms of cancer, comprising contacting the cellwith a therapeutically effective amount of the therapeutic construct ofany one of claims 1-39, or the composition of claim
 40. 45. A method oftreating a cell obtained from a subject exhibiting symptoms of cancer,comprising contacting cell ex vivo with a therapeutically effectiveamount of the therapeutic construct of any one of claims 1-39, or thecomposition of claim
 40. 46. The method of claim 44 or 45, wherein thecell is a cancer cell.
 47. The method of claim 44 or 45, wherein thecell is not a cancer cell.
 48. The method of claim 47, wherein the cellis an immune cell.
 49. The method of any one of claims 41-48, furthercomprising administering at least one treated cell back to a subject.50. A method of treating a subject diagnosed as having ahyperproliferative disease or condition, comprising administering to thesubject an effective amount of the composition of claim
 40. 51. Themethod of claim 50, wherein the hyperproliferative disease comprises oneor more of cancer, precancer, or cancer metastasis.
 52. The method ofclaim 50 or 51, wherein the hyperproliferative disease comprises one ormore of melanoma, lung cancer, breast cancer, pancreatic cancer, braincancer, prostate cancer, head and neck cancer, kidney cancer, colorectalcancer, lymphoma, colon cancer, or liver cancer.
 53. The method of anyone of claims 50-52, wherein the administering comprises one or more of:injection to or at a tumor in the subject; infusion locally to or at atumor in the subject; systemic injection in the subject; systemicinfusion in the subject; inhalation by the subject; oral administrationto the subject; or topical application to the subject.
 54. The method ofany one of claims 50-53, wherein the administering comprises microneedleapplication.
 55. A method of enhancing an effect of an anti-cancertherapy in a subject in need thereof, comprising administering to asubject in need thereof: an effective amount of the therapeuticconstruct of any one of claims 1-39, or the composition of claim 40; andat least one anti-cancer agent.
 56. The method of claim 55, wherein theanti-cancer agent is a chemotherapeutic agent, a targeted therapeuticagent, or an immune checkpoint inhibitor.
 57. The method of claim 55 or56, wherein the therapeutic construct or composition and the anti-cancertherapy are administered sequentially or concurrently.
 58. A method ofenhancing, increasing, or improving a radiation therapy effect in asubject diagnosed as having a neoplasia, comprising administering to asubject in need thereof: an effective amount of the therapeuticconstruct of any one of claims 1-39, or the composition of claim 40; andat least one radiation therapy.
 59. The method of claim 58, wherein thetherapeutic construct or composition and the radiation therapy areadministered sequentially or concurrently.
 60. The method of any one ofclaims 49-59, wherein the subject is human.
 61. A kit comprising: thetherapeutic construct of any one of claims 1-39; and at least oneanti-cancer agent.
 62. The kit of claim 61, wherein the anti-canceragent is a chemotherapeutic agent, a targeted therapeutic agent, or animmune checkpoint inhibitor.